Nephrol Dial Transplant (2014) 29: 228–231
doi: 10.1093/ndt/gft463
Advance Access publication 13 November 2013
A ray of light in the dark: alternative approaches to the
assessment and treatment of ischemic nephropathy
Naotake Tsuboi1, Shoichi Maruyama1, Seiichi Matsuo1 and Enyu Imai2
1
Department of Nephrology, Nagoya University Graduate School of Medicine, Nagoya, Aichi, Japan and 2Nakayamadera Imai Clinic,
Takarazuka, Hyogo, Japan
Correspondence and offprint requests to: Naotake Tsuboi; E-mail: tsubotake05@med.nagoya-u.ac.jp
Atherosclerotic renal arterial stenosis (ARAS) is a common
complication in elderly patients with chronic kidney disease
(CKD). It has been reported that 5–22% of elderly CKD
patients are affected by ARAS [1]. ARAS often causes severe
renovascular hypertension that is difficult to treat with antihy-
pertensive combination regimens. As angiotensin is generally
recognized to be responsible for renovascular hypertension, a
IN FOCUS
recent report has suggested that treatment with angiotensin-
converting enzyme (ACE) inhibitors or angiotensin receptor
blockers (ARB) is beneficial for RAS patients by leading to
lower cardiovascular event rates and reducing the requirement
for dialysis [2]. However, these benefits come with consider-
able risk for hospitalization for rapidly progressive renal
dysfunction in some ARAS population by excessive reduction
of glomerular filtration pressure [2, 3]. Moreover, ARAS is
associated with a high annual death rate of 16%, mainly due to
cardiovascular events [4], and is a predictor for death indepen-
dent of other conventional cardiovascular risk factors [5, 6].
ARAS is a clinical condition that is distressing to nephrologists
or interventional cardiologists engaged in treatment, because
the benefits of percutaneous transluminal renal angioplasty
(PTRA) and stenting of the renal arteries in ARAS patients
has been a topic of debate. A meta-analysis of three random-
ized trials [7–9] comparing conventional medication with
PTRA revealed that PTRA was not therapeutically beneficial to
blood pressure control in ARAS patients, even though it had a
significant effect in reducing the dosage of medication [10].
In focusing on renal deterioration, two large trials showed that
renal arterial stenting had no benefit over medical therapy
in outcomes relating to blood pressure, preservation of renal
function and mortality [11, 12]. Thus, there is yet no evidence
from clinical trials that revascularization reduces the incidence
of end-stage renal disease in patients with ARAS. A possible
reason for these negative findings is that some of the subjects
in the trials had no room for renal improvement by PTRA at
the time of entry.
© The Author 2013. Published by Oxford University Press on
behalf of ERA-EDTA. All rights reserved.
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Although insufficient efficacy of revascularization on renal
outcome in ARAS patients has been evidenced in several clini-
cal trials, there may be hope that revascularization improves
renal function in some populations of ARAS patients. Devel-
opment of an examination to evaluate renal function is needed
to determine which ARAS patients might benefit from revas-
cularization and subsequent follow-up management until
therapeutic intervention. The severity of RAS is assumed to be
a predictor for ischemic nephropathy, but it has been docu-
mented that severity of RAS only weakly relates to the presence
of ischemic nephropathy, as reported in a previous study
showing that the severity of proximal renal artery lesions was
often unassociated with renal dysfunction in ARAS patients
[13]. Thus, other feasible mechanisms beyond hemodynamic
decrement seem to influence the pathogenesis of RAS-mediated
parenchymal damage, and this should be considered in decid-
ing when and to whom therapeutic intervention should be
applied.
Lerman et al. [14–22] at the Mayo Clinic, Rochester, MN,
have consistently and intensively investigated the efficient
evaluation and treatment for post-stenotic kidneys in ARAS
by using immunological approaches. These investigators have
recently demonstrated the significance of elevated inflamma-
tory cytokines/chemokines, including interferon-gamma
(IFN-γ), tumor necrosis factor-alpha (TNF-α), monocyte che-
moattractant protein-1 (MCP-1) and interleukin (IL)-6, and
of CD68+ macrophage expansion for the evaluation of severity
of damage to the human post-stenotic kidney with ARAS [16,
19]. Lerman et al. also showed a strong association between
reduction of these elevated cytokines/chemokines in stenotic
kidneys and renal functional recovery after revascularization
in an experimental swine model of ARAS [15]. Therefore, it is
anticipated that treatments to modulate pro-inflammatory
cytokines/chemokines in post-stenotic kidneys can attenuate
renal dysfunction in post-stenotic kidneys and improve renal
prognosis after revascularization in the ARAS population.
228
Table 1. In vitro studies reporting MSC-mediated M2 macrophage polarization in co-culture experiments

MSC MSC MΦ origin M2MΦ Elevated MSC-derived Ref.

species source marker M2MΦ-associated key modulator
humoral factors for M2MΦ
polarization
Mouse Bone marrow C57BL/6 mouse Unverified IL-10, IL-12p40 PGE2 [29]
peritoneal MΦ
Human Gingiva Human peripheral CD206 IL-6, IL-10 GM-CSF, IL-6, [30]
blood monocyte PGE2
Human THP-1 cell
Human Bone marrow NOD/SCID mouse CD206, MHCII, TGF-β, IL-10 IL-10 [31]
CD163, Arg-1
Human Bone marrow Human peripheral CD206 IL-10 unverified [32]
blood monocyte
Human Bone marrow Mouse J774A cell CD206 IL-1RA, IL-10 PGE2 [33]
Human Adipose Human peripheral CD206, CD163, IL-10, VEGF IL-4. IL13 [34]
blood monocyte CD16

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Wister-Kyoto Adipose Wister-Kyoto rat CD163, CD206 IL-10 IL-6, PGE2 [35]
rat peritoneal MΦ
MΦ, macrophage; PGE2, prostaglandin E2, GM-CSF, granulocyte macrophage colony-stimulating factor; MHCII, major histocompatibility complex II; Arg-1, arginase-1; TGF-β,
transforming growth factor β, IL-1RA, interleukin-1 receptor antagonist; VEGF, vascular endothelial growth factor.

Table 2. In vivo studies reporting MSC-mediated M2 macrophage polarization in animal disease models with MSC treatment

MSC species MSC Treatment Model Target M2MΦ Elevated Decreased Ref.
source animal disease marker tissue tissue
humoral humoral
factors factors
Human Gingiva MSC i.v. C57BL/6 mouse Skin wound healing Arg-1; IL-10 TNF-α, IL-6 [30]

IN FOCUS
RELM-α
Human Bone marrow MSC i.v. NOD/SCID Acute myocardial CD206 IL-10 IL-1β, IL-6 [31]
mouse infarction
Human Bone marrow MSC i.l. Sprague–Dawley Spinal cord injury Arg-1, CD206 IL-4, IL-13 TNF-α, IL-6 [36]
rat
C57BL/6 mouse Bone marrow MSC i.t. CM i. C57BL/6 mouse LPS-induced ALI Ym1 IGF-1a Unverified [37]
t.
Wister–Kyoto rat Adipose MSC i.v. Wister–Kyoto rat Anti-GBM GN CD163 IL-10 IL-1β, [35]
CD206 IL-12p70
Sprague–Dawley rat Fetal membrane, bone MSC sheet Lewis rat Acute myocardial CD163 Unverified Unverified [38]
Lewis rat marrow infarction
MΦ, macrophage; i.v., injected intravenously; Arg-1, arginase-1; RELM-α, resistin-like molecule-α; i.l., injected locally; i.t., injected intratracheally; CM, conditional media; ALI, acute
lung injury; Ym1, secretory protein Ym1; IGF-1, type 1 insulin-like growth factor; anti-GBM GN, glomerular basement membrane glomerulonephritis.
a
Result in CM administration.

Systemic administration of neutralizing antibodies or antago-
nists for individual inflammatory cytokines/chemokines or for
their receptors can be offered to candidates to attenuate post-
stenotic renal dysfunction. However, various types of leukocytes
and resident renal cells locally secrete these inflammatory
mediators, orchestrating the development of renal inflam-
mation and the subsequent fibrosis at the sites of injury
[21, 23]. Therefore, therapeutic approaches to exert pleiotropic
immunomodulation specific for inflammation in ARAS kidneys
are desirable. In an experimental swine model of RAS, several
investigators have exploited the intrarenal administration of
mesenchymal stem cells (MSCs) from adipose tissue to modu-
late renal inflammation [14, 17, 22]. In several animal models
of acute kidney injury, including acute ischemic nephropathy
and cisplatin-induced nephropathy, MSC from bone marrow,
adipose tissue or the umbilical cord have recently become
substantial options for cell-based therapy through their power-
ful immunomodulatory activity [24, 25]. Also, in recent
studies by Lerman et al., the renoprotective effect of MSC
transfer was clearly demonstrated in an experimental swine
ARAS model. The intrarenal administration of MSC, in
addition to revascularization by PTRA and stenting, signifi-
cantly improved the glomerular filtration rate (GFR), renal
blood flow (RBF), interstitial fibrosis, inflammation, microvas-
cular rarefaction and oxidative stress in stenotic kidneys com-
pared with ARAS and ARAS + PTRA [14, 17]. It is of note
that PTRA alone did not show a beneficial effect against
functional and pathological damage despite normalization of
the mean arterial pressure in stenotic kidneys, which corrobo-
rates the negative outcome demonstrated in several clinical trials
for PTRA in human ARAS patients. Moreover, even without re-
vascularization, MSC significantly attenuated GFR and RBF,

229 A ray of light in the dark

 which was associated with improvement with respect to renal
inflammation, endoplasmic reticulum stress and apoptosis in
swine ARAS kidneys [22]. This indicated that the renoprotecive
effect of administered MSC is independent of normalization for
renal arterial stenosis.
In this issue of Nephrology Dialysis Transplantation,
Eirin et al. externally validated renal inflammation in post-
stenotic kidneys in swine models of RAS. These authors
showed that proinflammatory cytokine/chemokine levels, in-
cluding IFNγ, TNFα and MCP-1, both in the renal veins and
the renal tissues of RAS kidneys, were significantly decreased,
but the anti-inflammatory cytokine IL-10 was elevated in MSC-
-treated animals compared with RAS animals without MSC
treatment. As the cytokine/chemokine levels in the renal veins
directly reflect the degree of organ inflammation in RAS kid-
neys, intravenous cytokine/chemokine sampling can be a strong
tool for evaluating renal damage and therapeutic efficacy.
In addition to strong correlation of the cytokine/chemokine
profile in the renal veins with their tissue expression in RAS
kidneys, it was interesting that the profile was also associated
with macrophage subpopulations among experimental groups
of control, RAS animals and MSC-treated RAS animals.
Macrophages are currently defined as heterogeneous popu-
lations of classically activated macrophages (M1 macrophages)
and alternatively activated macrophages (M2 macrophages),
which include further subpopulations of M2a, M2b and M2c
cells classified by specific markers and functions [26–28]. Renal
injury rapidly promotes ‘inflammatory’ M1 macrophages, and
IN FOCUS
then this response is shifted into ‘anti-inflammatory’ M2
macrophages according to their microenvironment. The
response of M1 macrophages induces renal damage, whereas
M2 macrophages have been demonstrated to have an anti-
inflammatory property, leading to resolution of inflammation,
tissue regeneration and repair and subsequent tissue fibrosis
[26]. Recently, several in vitro and in vivo studies have demon-
strated the predominant potential of MSC or MSC-derived
conditional media to promote phenotypic change of macro-
phages to M2 cells (Tables 1 and 2). Also in this study, the
authors found predominant infiltration of M2 macrophages,
in particular CD163+/CD206+/Fizz1-M2 macrophages (M2c),
into RAS + MSC kidneys despite a comparable number of M1
cells that infiltrated into RAS kidneys. These data strongly sug-
gest that transferred MSCs exert a renoprotective function
through efficient macrophage conversion into M2 cells. This
was further evidenced by the results from an in vitro co-culture
system with MSC and human monocytes, where porcine MSC
directly polarized human monocytes into arginase-1-expressing
M2 cells.
For further understanding of the precise mechanism of
MSC-mediated M2 macrophage polarization, we must wait
for further investigation in animal models, including investi-
gation of M2 macrophage transfer into RAS kidneys. More-
over, it should be noted that M2 macrophage polarization
might be one of the representative MSC functions, because
MSC can affect other leukocyte subsets and renal resident
cells by their paracrine effect with great tropism in damaged
organs. With respect to clinical application of MSC transfer,
there are still some issues in need of resolution, including cell
N. Tsuboi et al.
source, rejection, carcinogenesis, injection route and other
adverse effects common in stem cell therapies. However, we
can find hope for improved prognosis for ARAS patients from
the forthcoming results of the current study by Eirin et al.
This study offers two major exciting prospects: one is the
potentially beneficial cytokine/chemokine profile of renal veins
for the assessment of parenchymal inflammation in ARAS
and the other is the greater property of MSC as a therapeutic
option of cell therapy beyond revascularization for ARAS.
AC K N O W L E D G E M E N T S
This work was supported by a Grant-in-Aid for Scientific
Research (B, Se.M.) and (C, N.T. and Sh.M.) from the Ministry
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of Education, Culture, Sports, Science and Technology and the
Japan Society for the Promotion of Science (JSPS), a Grant-in-
Aid for Progressive Renal Diseases Research, Research on
Intractable Disease, from the Ministry of Health, Labour and
Welfare of Japan (N.T., Sh.M. and Se.M.).
C O N F L I C T O F I N T E R E S T S TAT E M E N T
The authors have no conflicts of interest to disclose.
(See related article by Eirin et al. Renal vein cytokine release as
an index of renal parenchymal inflammation in chronic exper-
imental renal artery stenosis. Nephrol Dial Transplant 2014;
29: 274–282.)
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A ray of light in the dark