Low-dose carbon monoxide inhibits progressive chronic allograft nephropathy
and restores renal allograft function
Atsunori Nakao,1Gaetano Faleo,1Michael A. Nalesnik,2Joao Seda-Neto,1Junichi Kohmoto,1
and Noriko Murase1
Departments of1Surgery and2Pathology, Thomas E. Starzl Transplantation Institute, University of Pittsburgh Medical
Center, Pittsburgh, Pennsylvania
Submitted 2 December 2008; accepted in final form 14 April 2009
Nakao A, Faleo G, Nalesnik MA, Seda-Neto J, Kohmoto J,
Murase N. Low-dose carbon monoxide inhibits progressive chronic
allograft nephropathy and restores renal allograft function. Am J
Physiol Renal Physiol 297: F19–F26, 2009. First published April 15,
2009; doi:10.1152/ajprenal.90728.2008.—Chronic allograft nephrop-
athy (CAN) represents progressive deterioration of renal allograft
function with fibroinflammatory changes. CAN, recently reclassified
as interstitial fibrosis (IF) and tubular atrophy (TA) with no known
specific etiology, is a major cause of late renal allograft loss and
remains a significant deleterious factor of successful renal transplan-
tation. Carbon monoxide (CO), an effector byproduct of heme oxy-
genase pathway, is known to have potent anti-inflammatory and
antifibrotic functions. We hypothesized that inhaled CO would inhibit
fibroinflammatory process of CAN and restore renal allograft func-
tion, even when the treatment was initiated after CAN was estab-
lished. Lewis rat kidney grafts were orthotopically transplanted into
binephrectomized allogenic Brown Norway rats under brief tacroli-
mus (0.5 mg/kg im, days 0–6). At day 60, CO (20 ppm) inhalation
was initiated to recipients and continued until day 150 or animal
death. Development of CAN was confirmed at day 60 with decreased
creatinine clearance (CCr), significant proteinuria, and histopatholog-
ical findings of TA, IF, and intimal arteritis. Air-treated control
recipients continued to deteriorate with further declines of CCr and
increases of urinary protein excretion and died with a median survival
of 82 days. In contrast, progression of CAN was decelerated when
recipients received CO on days 60–150, showing markedly improved
graft histopathology, restored renal function, and improved recipient
survival to a median of ?150 days. CO significantly reduced intra-
graft mRNA levels for IFN-? and TNF-? at day 90. Expression of
profibrotic TGF-?/Smad was significantly suppressed with CO, to-
gether with downregulation of ERK-MAPK pathways. Continuous
CO (20 ppm) treatment for days 0–30, days 30–60, or days 0–90, or
daily 1-h CO (250 ppm) treatment for days 0–90, also showed
efficacy in inhibiting CAN. The study demonstrates that CO is able to
inhibit progression of fibroinflammatory process of CAN, restore
renal allograft function, and improve survival even when the treatment
is started after CAN is diagnosed.
kidney transplantation; renal fibrosis; anti-inflammatory effects; anti-
ALTHOUGH THE FREQUENCY and severity of acute rejection epi-
sodes have been significantly improved, the half-life of renal
allografts has stayed unchanged for over a decade (2, 31).
There is a gradual loss of ?5% renal allografts per year,
resulting in 5- and 10-yr graft survival rates of ?70 and 50%,
respectively (7). The most common clinical correlate of late
allograft loss is referred to nonspecifically as chronic allograft
nephropathy (CAN), and more than 40% of kidney allograft
recipients suffer from CAN during 5 yr after transplantation (3,
7). CAN is characterized by progressive renal allograft dys-
function with histopathological features of chronic interstitial
fibrosis (IF), tubular atrophy (TA), vascular occlusive changes,
and glomerulosclerosis. Thus, CAN is a clinicopathological
classification caused by multiple factors, including alloantigen-
dependent immune responses, as well as nonimmunological
factors, such as ischemia-reperfusion (I/R) injury, calcineurin
inhibitor nephrotoxicity, metabolic and cardiovascular disor-
ders, and exacerbating preexisting donor disease (10, 22, 28).
This has led the Banff group (26) to recommend eliminating
the term CAN in those cases with no known etiology in favor
of “interstitial fibrosis and tubular atrophy with no known
specific cause” (IF/TA). However, since the term CAN is
presently entrenched in the literature, it will be used as a
synonym for IF/TA in its descriptive, generic sense in this
report and is not meant to imply any specific etiology. There
have been a number of approaches aiming at reducing the
impact of CAN; however, no practical therapeutic strategies
are currently available to prevent and treat CAN.
Recent studies showed that endogenously generated carbon
monoxide (CO) by heme oxygenase (HO) system serves as a
key mechanism to maintain the integrity of the physiological
function of organs (15, 30). Numerous reports subsequently
show that exogenously delivered CO provides cytoprotection
in various injury conditions. These observations strongly indi-
cate that CO is a biologically active gaseous molecule in the
body (16). Earlier study in our laboratory demonstrated that
treatment of kidney allograft recipients with inhaled low-dose
CO (20 ppm) for the first 30 days of kidney transplant (KTx)
effectively prevented CAN development by 1) inhibiting
recruitment of inflammatory infiltrates, 2) downregulating
proinflammatory mediators, cytokines, and chemokines,
3) promoting vasodilation and maintaining cortical blood flow,
4) preventing tissue fibrosis, and 5) regulating T cell alloreac-
Considering potent cytoprotective functions of CO observed
in our previous study, we hypothesized in this study that CO
could reverse established CAN and restore allograft function
even when CO was given after CAN was developed. To test
the hypothesis, rat kidney allograft recipients were kept with-
out treatment for the first 60 days of KTx to develop CAN and
then treated with 20 ppm CO. The result shows that low-dose
CO is able to reverse CAN and restore renal allograft function
through its anti-inflammatory and antifibrosis actions.
Address for reprint requests and other correspondence: N. Murase, Thomas
E. Starzl Transplantation Institute, Dept. of Surgery, E1555 Biomedical Sci-
ence Tower, Univ. of Pittsburgh, Pittsburgh, PA 15213 (e-mail: murase
Am J Physiol Renal Physiol 297: F19–F26, 2009.
First published April 15, 2009; doi:10.1152/ajprenal.90728.2008.
0363-6127/09 $8.00 Copyright © 2009 the American Physiological Society http://www.ajprenal.orgF19
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REVERSAL OF CHRONIC ALLOGRAFT NEPHROPATHY WITH CO
AJP-Renal Physiol • VOL 297 • JULY 2009 • www.ajprenal.org