Am J Physiol Renal Physiol 301: F436–F442, 2011.
First published April 27, 2011; doi:10.1152/ajprenal.00162.2011.
Genetic deficiency of Smad3 protects against murine ischemic acute
kidney injury
Karl A. Nath,1* Anthony J. Croatt,1 Gina M. Warner,2 and Joseph P. Grande2*
1
Division of Nephrology and Hypertension and 2Department of Pathology, Mayo Clinic, Rochester, Minnesota
Submitted 22 March 2011; accepted in final form 25 April 2011
IL-6; TGF-␤1; heme oxygenase-1; chronic kidney disease
TRANSFORMING GROWTH FACTOR-␤1 (TGF-␤1) is a pleiotropic
cytokine that affects many of the major pathobiological processes that contribute to tissue injury (4, 7, 8, 18, 30, 33, 40, 42,
49). Such effects of TGF-␤1 are commonly cell specific and
context dependent (18, 33); for example, depending upon the
experimental conditions, TGF-␤1 may promote processes that
are either antiapoptotic or proapoptotic and may exert actions
that are either proinflammatory or anti-inflammatory. Such
diverse and, at times, seemingly contradictory effects of
TGF-␤1 probably reflect the myriad signaling systems recruited by TGF-␤1, and these include 1) the Smad family of
proteins, 2) the MAPK system, 3) phosphatidylinositol 3-kinase signaling, 4) small GTPases, 5) mammalian target of
rapamycin, and 6) ILK (4, 7, 8, 18, 30, 33, 40, 42, 49).
However, whatever the divergent, cell-specific effects of
TGF-␤1 on biological processes, one effect of TGF-␤1 is
firmly and consistently established irrespective of the involved
* K. A. Nath and J. P. Grande contributed equally as senior authors on this
paper.
Address for reprint requests and other correspondence: J. P. Grande, Mayo
Clinic, Medical Sciences Bldg. 2_66, 200 First St. SW, Rochester, MN 55905
(e-mail: [email protected]).
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tissues: the capacity of TGF-␤1 to promote matrix expansion
and fibrogenesis, processes in which the signaling intermediate, Smad3, is a fundamental participant. Indeed, TGF-␤1 is
uniformly regarded as a key contributor to the progression of
chronic kidney disease, a contribution that reflects, at least in
part, the signaling effects of Smad3 (4, 7, 8, 30, 40, 42, 49, 51).
The pathobiological significance of TGF-␤1 in acute kidney
injury is increasingly of interest (2, 3, 14, 15, 26 –28, 45). Early
and marked upregulation of TGF-␤1 and its receptors occurs
following acute renal ischemia (2, 3, 14, 45), and, as shown by
our prior studies, TGF-␤1 is markedly induced in a sustained
fashion following heme protein-induced renal injury (36).
Studies in the rat model of acute renal ischemia demonstrate
that antagonism of the actions of TGF-␤1 inhibits the synthesis
of extracellular matrix proteins, but fails to influence the course
of renal function following such ischemic injury (14, 45).
However, in other models of acute ischemic injury, a cytoprotective role for TGF-␤1 has been demonstrated. In a murine
model of acute renal ischemia, a deficiency in TGF-␤1 exacerbates renal dysfunction and worsens renal histological injury
(15), and in in vitro models inhibition of TGF-␤1 exacerbates
oxidant-induced necrosis of proximal tubular epithelial cells
(27). Acquired resistance to renal ischemic injury in certain
settings is also TGF-␤1 dependent. For example, prior exposure to the anesthetic agent sevoflurane protects against renal
damage following acute ischemic insults, and such protection
by sevoflurane is impaired either by the administration of a
neutralizing TGF-␤1 antibody, or in TGF-␤1⫹/⫺ mice (26). In
studies in vitro, a neutralizing TGF-␤1 antibody vitiates the
cytoprotection conferred by sevoflurane against hydrogen peroxide-induced injury to proximal tubular epithelial cells (27).
In light of evidence that TGF-␤1 is protective in acute
kidney injury, and the fundamental role of Smad3 in promoting
the injurious effects of TGF-␤1, at least in chronic kidney
disease, the present study was undertaken to examine the role
of Smad3 in ischemic acute kidney injury.
MATERIALS AND METHODS
Murine Smad3⫺/⫺ model. Smad3⫹/⫹ and Smad3⫺/⫺ mice employed for the present studies were generated from colonies of mice
established from breeder stock obtained from the Jackson Laboratory
(129-Smad3tm1Par/J, stock no. 003451, Bar Harbor, ME), and maintained by mating Smad3⫹/⫺ males with Smad3⫹/⫺ females. Offspring
were genotyped at the time of weaning using PCR to amplify the
wild-type and mutant alleles of genomic DNA from tail samples. For
studies of Smad3⫺/⫺ and Smad3⫹/⫹ mice, age-matched male mice
from 10 to 24 wk were employed. Additionally, in other studies
14-wk-old male C57BL6J mice (Jackson Laboratory) were employed.
All experiments were performed in accordance with the guidelines of
the National Institutes of Health and approved by the Institutional
Animal Care and Use Committee of the Mayo Clinic.
1931-857X/11 Copyright © 2011 the American Physiological Society
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Nath KA, Croatt AJ, Warner GM, Grande JP. Genetic deficiency
of Smad3 protects against murine ischemic acute kidney injury. Am J
Physiol Renal Physiol 301: F436 –F442, 2011. First published April 27,
2011; doi:10.1152/ajprenal.00162.2011.—TGF-␤1 contributes to
chronic kidney disease, at least in part, via Smad3. TGF-␤1 is induced
in the kidney following acute ischemia, and there is increasing
evidence that TGF-␤1 may protect against acute kidney injury. As
there is a paucity of information regarding the functional significance
of Smad3 in acute kidney injury, the present study explored this issue
in a murine model of ischemic acute kidney injury in Smad3⫹/⫹ and
Smad3⫺/⫺ mice. We demonstrate that, at 24 h after ischemia, Smad3
is significantly induced in Smad3⫹/⫹ mice, whereas Smad3⫺/⫺ mice
fail to express this protein in the kidney in either the sham or
postischemic groups. Compared with Smad3⫹/⫹ mice, and 24 h
following ischemia, Smad3⫺/⫺ mice exhibited greater preservation of
renal function as measured by blood urea nitrogen (BUN) and serum
creatinine; less histological injury assessed by both semiquantitative
and qualitative analyses; markedly suppressed renal expression of
IL-6 and endothelin-1 mRNA (but comparable expression of MCP-1,
TNF-␣, and heme oxygenase-1 mRNA); and no increase in plasma
IL-6 levels, the latter increasing approximately sixfold in postischemic Smad3⫹/⫹ mice. We conclude that genetic deficiency of Smad3
confers structural and functional protection against acute ischemic
injury to the kidney. We speculate that these effects may be mediated
through suppression of IL-6 production. Finally, we suggest that
upregulation of Smad3 after an ischemic insult may contribute to the
increased risk for chronic kidney disease that occurs after acute renal
ischemia.
Smad3 DEFICIENCY PROTECTS AGAINST MURINE AKI
F437
Fig. 1. Western blot analysis of Smad3 expression in
kidneys of Smad3⫹/⫹ mice 24 h after bilateral renal
ischemia (IR) for 25 min or sham surgery (Sham). Each
lane represents protein extracted from a single kidney
of an individual mouse. Equivalency of protein loading
was assessed by immunoblotting for ␤-actin, and individual and mean standardized densitometric values are
provided below the Western blot analysis. Smad3 expression was significantly higher in mice subjected to
IR compared with mice subjected to Sham (P ⬍ 0.05).
␤-actin (catalog no. 612657, BD Biosciences, San Jose, CA) were
used in overnight incubations at 4°C. Horseradish peroxidase-conjugated secondary antibodies were then used, and bands were visualized
using an enhanced chemiluminescence method.
Serum IL-6 quantitation by ELISA. Serum concentrations of IL-6
protein were measured using a commercially available ELISA set
(OptEIA, catalog no. 555240, BD Biosciences) according to the
manufacturer’s assay instructions.
Histological analysis. Histological analysis was performed on
5-␮m hematoxylin- and eosin-stained sections prepared from formalin-fixed, paraffin-embedded renal tissues from Smad3⫹/⫹ and
Smad3⫺/⫺ mice 24 h after bilateral renal ischemia or sham operation.
Blinded semiquantitative evaluation of necrosis was performed by
assessing the percentage of tubules demonstrating any evidence of
epithelial cell necrosis, and the degree of extension of necrosis into the
cortex, determined by the mean percentage of the distance from the
corticomedullary junction to the surface of the kidney in which
necrosis was present.
Statistics. Data are expressed as means ⫾ SE. Data for Smad3⫺/⫺
and Smad3⫹/⫹ mice for a given condition were compared using
Student’s t-test for parametric data and the Mann-Whitney test for
nonparametric data. Results were considered significant at P ⬍ 0.05.
RESULTS
Effect of renal ischemia on Smad3 expression. At 3 h,
Smad3 expression by Western blot analysis was unaltered in
response to renal ischemia compared with sham ischemia in
wild-type mice (data not shown). However, at a later time
point, 24 h, there was a marked and significantly increased
expression (⬃3-fold) of Smad3 in Smad3⫹/⫹ mice subjected to
renal ischemia (Fig. 1). Western blot analysis also confirmed
that Smad3⫺/⫺ mice failed to express Smad3 in the kidney
after either sham ischemia or renal ischemia, thereby confirming the deficiency of this protein in this mutant strain (Fig. 2).
Effect of Smad3 deficiency on renal function. Renal functional studies were undertaken in two separate protocols, employing different durations of ischemia that lead to durationdependent, renal dysfunction in wild-type mice. As demonstrated, Smad3⫺/⫺ mice exhibited substantial preservation in
renal function in response to renal ischemia as reflected by
Fig. 2. Western blot analysis of Smad3 expression in
kidneys of Smad3⫹/⫹ and Smad3⫺/⫺ mice 24 h after IR
for 25 min or Sham. Each lane represents protein
extracted from a single kidney of an individual mouse.
Equivalency of protein loading was assessed by immunoblotting for ␤-actin, and individual and mean standardized densitometric values are provided below the
Western blot analysis. No expression of Smad3 occurred in Smad3⫺/⫺ mice.
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Murine model of renal ischemia-reperfusion. This model of acute
renal ischemia was induced in mice as described in detail by our prior
studies (20, 21, 37, 39, 47). Briefly, after mice were anesthetized with
pentobarbital (50 mg/kg ip), the renal pedicles were gently dissected
and bilateral renal ischemia was induced with nontraumatic clamps
(RS5426, Micro Aneurysm clip, straight, 10 mm, 125-g pressure;
Roboz Surgical Instruments, Rockville, MD) through a midline abdominal incision. Two protocols of renal ischemia consisting of either
22.5 or 25 min were separately employed in Smad3⫹/⫹ and
Smad3⫺/⫺ mice so as to determine the consistency of the observed
effect of the deficiency of Smad3 on different durations of renal
ischemia. Sham procedures of the duration of the 25-min ischemia
procedure included the abdominal incision but excluded renal pedicle
dissection and clamping. For these studies, renal function was assessed by the measurement of serum creatinine and blood urea
nitrogen (BUN) levels at 24 h after ischemia using a Creatinine
Analyzer 2 and a BUN Analyzer 2 (Beckman Instruments, Fullerton, CA).
mRNA expression by quantitative real-time RT-PCR. For analysis
of gene expression, total RNA was extracted from snap-frozen mouse
renal tissues using the TRIzol method (Invitrogen, Carlsbad, CA) and
subsequently further purified with an RNeasy Mini kit (Qiagen,
Valencia, CA), according to each manufacturer’s protocol and as
described in detail in our prior studies (39, 48). Three hundred
nanograms of purified total RNA were used in 30-␮l reverse transcription reactions (Transcriptor First Strand cDNA Synthesis kit,
Roche Applied Science, Indianapolis, IN) employing random hexamers. The resulting cDNA was used in quantitative real-time PCR
analysis as in our earlier study (39). Reactions were performed on an
ABI Prism 7900HT (Applied Biosystems, Foster City, CA) using
TaqMan Mastermix reagent (part no. 4324020, Applied Biosystems).
Probes and primers obtained as assay sets (TaqMan Gene Expression
Assays, Applied Biosystems) were employed in these reactions according to the manufacturer’s protocol. Parameters for quantitative
PCR were as follows: 10 min at 95°C, followed by 40 cycles of
amplification for 15 s at 95°C, and 1 min at 60°C. Expression of 18S
rRNA was used for standardization of the expression of each target
gene.
Western blot analysis. Western blot analysis was performed as
described in our previous studies (39, 48). Briefly, proteins (150 ␮g)
were separated on 10% Tris-HCl gels (Bio-Rad) and transferred to
polyvinylidene fluoride membranes. Primary antibodies for Smad3
(catalog no. 9523, Cell Signaling Technology, Danvers, MA) and
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Smad3 DEFICIENCY PROTECTS AGAINST MURINE AKI
DISCUSSION
BUN (Fig. 3) and serum creatinine (Fig. 4) 24 h after renal
ischemia.
Effect of Smad3 deficiency on renal histological injury.
Along with this preservation of renal function in Smad3⫺/⫺
mice following renal ischemia, there was substantial reduction
in renal histological injury as reflected by less acute tubular
necrosis, and less intratubular sloughing and cast formation in
Smad3⫺/⫺ mice following renal ischemia (Fig. 5). Semiquantitative analyses confirmed such qualitative analyses:
scores for the severity of acute tubular necrosis and the
extent to which tubular necrosis extended from the corticomedullary junction into the cortex were both significantly
lower in Smad3⫺/⫺ mice following renal ischemia either
22.5 or 25 min in duration (Fig. 6).
Effect of Smad3 deficiency on renal gene expression. In an
attempt to determine the basis for this effect of Smad3 deficiency, we assessed the expression of cytokines that contribute
to ischemic acute kidney injury. As previously described,
expression of IL-6 mRNA is markedly increased after renal
ischemia, and this effect was greatly attenuated in Smad3⫺/⫺
mice compared with Smad3⫹/⫹ mice following renal ischemia
(Fig. 7). While not as striking, expression of endothelin-1 was
also significantly reduced in Smad3⫺/⫺ mice compared with
Smad3⫹/⫹ mice following ischemia (Fig. 8). Interestingly, a
similar pattern was found with TGF-␤1 (Fig. 9). Other cytokines that are well established as contributors to ischemic acute
kidney injury, specifically, MCP-1 and TNF-␣, were not significantly altered by Smad3 deficiency (Table 1). In addition to
pathways that promote renal injury, we also examined a pathway that can protect against renal injury, namely, heme oxygenase (HO)-1 (19, 35): HO-1 mRNA expression was comparably induced, thus demonstrating that this was unlikely to be
a mechanism contributing to the protective effects of Smad3
deficiency (Table 1).
Effect of Smad3 deficiency on plasma IL-6 levels. We also
measured plasma levels of IL-6, since systemic levels of
cytokines are a determinant of the outcome from acute kidney
injury. Following ischemia, IL-6 was markedly increased at 24
h in Smad3⫹/⫹ mice, and such elevation in IL-6 after ischemia
did not occur in Smad3⫺/⫺ mice (Fig. 10).
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Fig. 4. Serum creatinine levels 24 h after IR for either 22.5 or 25 min in
duration and after Sham in Smad3⫹/⫹ and Smad3⫺/⫺ mice; n ⫽ 7– 8/group.
*P ⬍ 0.05, Smad3⫺/⫺ vs. Smad3⫹/⫹ mice subjected to respective IR duration.
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Fig. 3. Serum blood urea nitrogen (BUN) levels 24 h after IR for either 22.5
or 25 min in duration and after Sham in Smad3⫹/⫹ and Smad3⫺/⫺ mice; n ⫽
7– 8/group. *P ⬍ 0.05, Smad3⫺/⫺ vs. Smad3⫹/⫹ mice subjected to respective
IR duration.
Our data, in aggregate, demonstrate that Smad3 is induced in
the kidney following acute renal ischemia and that genetic
deficiency of Smad3 leads to greater preservation of renal
function, less histological injury, reduced renal expression of
specific cytokines, and less systemic inflammation following
acute renal ischemia. Based on these findings, we conclude that
induction of Smad3 is a maladaptive response that contributes
to the functional and structural damage attendant upon acute
renal ischemia.
In an attempt to uncover mechanisms that may underlie the
reduced ischemic injury incurred in Smad3⫺/⫺ mice, we evaluated the expression of a number of cytokines that contribute
to acute kidney injury following ischemia. The most dramatic
effect was noted in the renal expression of IL-6, the latter
markedly lower in Smad3⫺/⫺ mice compared with Smad3⫹/⫹
mice following ischemia. These findings are of interest in that
multiple lines of evidence attest to the injurious effects of IL-6
following an ischemic insult. For example, the administration
of a neutralizing IL-6 antibody leads to greater preservation of
renal function and less histological injury following acute renal
ischemic injury (22, 38); IL-6⫺/⫺ mice evince less renal
dysfunction and histological injury in response to an acute
ischemic insult (22, 38); HO-1⫺/⫺ mice, compared with HO1⫹/⫹ mice, exhibit an exaggerated induction of IL-6, increased
renal dysfunction, and increased mortality following renal
ischemia (47), while the administration of a neutralizing IL-6
antibody attenuates such renal dysfunction and mortality observed in HO-1⫺/⫺ mice following ischemia (47). Clinical
observations support the pathogenetic significance of increased
IL-6 production as increased urinary excretion of IL-6 is a
predictor for human acute kidney injury (9, 25). Our finding
that IL-6 mRNA was markedly induced in the ischemic kidney,
and that such expression of IL-6 was drastically reduced in the
ischemic kidney in Smad3⫺/⫺ mice, raises the possibility that
the protection conferred by Smad3 deficiency may reflect
reduced production of IL-6. In this regard, observations in
other cell types demonstrate that the induction of IL-6 by
TGF-␤1 critically requires Smad3 (1, 13).
Smad3 DEFICIENCY PROTECTS AGAINST MURINE AKI
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Renal ischemia provokes a systemic inflammatory response,
and among the recognized and prominent cytokines so produced is IL-6. Such a systemic response is implicated in the
adverse distant effects of renal ischemia such as lung injury
(23). Clinical observations in acutely ill patients demonstrate
that plasma IL-6 levels predict ventilator dependency, severity
of acute kidney injury, and mortality that occur in this patient
population (6, 29, 44). Our data demonstrate that the marked
elevation in plasma IL-6 levels observed following ischemia in
wild-type mice did not occur in Smad3⫺/⫺ mice. Thus the
suppressive effect of the deficiency of Smad3 on IL-6 occurs
not only regionally in the kidney but also in the systemic
circulation.
It is possible that the protective effect of Smad3 deficiency
we observed may reflect decreased production of other cytokines that are recognized as contributors to acute ischemic
injury; in this regard, substantial evidence indicates that renal
injury following acute ischemia can reflect increased renal
expression of endothelin-1, MCP-1, and TNF-␣ (10 –12, 17,
24, 31, 34, 50). Our data demonstrate that Smad3⫺/⫺ mice
compared with Smad3⫹/⫹ mice, in response to ischemia, had a
blunted expression of ET-1 mRNA, and thus such reduced
expression in ET-1 may be relevant to the reduced renal injury
in Smad3⫺/⫺ mice observed after ischemia. These findings are
consistent with observations that cellular induction of ET-1 by
TGF-␤1 requires Smad3 (5, 41).
The finding that MCP-1 and TNF-␣ expression was comparably increased in the kidney in Smad3⫹/⫹ and Smad3⫺/⫺ mice
is of interest for at least two reasons. First, it is unlikely that the
adverse effects of Smad3 in acute renal ischemia are mediated
through these cytokines; second, the lack of alteration in
expression of these cytokines indicates that the reduction in
IL-6 and ET-1 was not a nonspecific, generalized reductive
effect on cytokine expression attendant upon the deficiency of
Smad3.
We observed that renal induction of TGF-␤1 mRNA was
substantially reduced in Smad3-deficient mice following
ischemia. To the best of our knowledge, we are unaware of
Fig. 6. Semiquantitative assessment of necrosis in the kidney 24 h after IR for either 22.5 or
25 min in duration in Smad3⫹/⫹ and
Smad3⫺/⫺ mice. Shown are blinded analyses
of the percentage of tubules exhibiting evidence of epithelial cell necrosis (A) and extension of cortical necrosis (B), the latter determined by the mean percentage of the distance
from the corticomedullary junction to the surface of the kidney in which necrosis was present. No necrosis was observed in the kidneys
of Smad3⫹/⫹ and Smad3⫺/⫺ mice subjected to
Sham (data not shown). *P ⬍ 0.05, Smad3⫺/⫺
vs. Smad3⫹/⫹ mice subjected to respective IR
duration.
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Fig. 5. Histological examination of the kidney after
IR in Smad3⫹/⫹ and Smad3⫺/⫺ mice. Lower power
(⫻100, A and B) and higher power (⫻200, C and D)
views of the kidney in Smad3⫹/⫹ (A and C) and
Smad3⫺/⫺ (B and D) mice 24 h after IR for 25 min
are shown. Smad3⫺/⫺ mice exhibited less renal
injury compared with Smad3⫹/⫹ mice in response to
this duration of ischemia. No histological injury was
observed in the kidneys of Smad3⫹/⫹ and
Smad3⫺/⫺ mice subjected to Sham (not shown).
Tissue sections were stained with hematoxylin and
eosin.
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Smad3 DEFICIENCY PROTECTS AGAINST MURINE AKI
studies examining tissue expression of TGF-␤1 in Smad3deficient mice following an imposed stress in vivo. While
the basis for this reduced TGF-␤1 mRNA expression in
stressed Smad3-deficient mice is uncertain, we speculate
that a positive feedback loop exists between TGF-␤1 and
Smad3 in the ischemic kidney: in response to ischemia,
TGF-␤1 induces Smad3, and induced Smad3, in turn, fosters
TGF-␤1 expression.
Our finding that Smad3, a signaling molecule downstream of
TGF-␤1, contributes to ischemic acute kidney injury merits
discussion within the context of TGF-␤1 as a cytokine that
protects against ischemic acute kidney injury (15, 26 –28).
TGF-␤1 is pleiotropic in its actions and engages diverse
signaling molecules. Even within the Smad family, constituent
members exert divergent effects. For example, quite recently it
has been shown that Smad2 offsets the fibrogenic actions of
Fig. 8. Renal expression of ET-1 mRNA 24 h after IR for either 22.5 or 25 min
in duration and after Sham in Smad3⫹/⫹ and Smad3⫺/⫺ mice. ET-1 mRNA
expression was determined by quantitative real-time RT-PCR and standardized
for 18S rRNA;. n ⫽ 7– 8/group. *P ⬍ 0.05, Smad3⫺/⫺ vs. Smad3⫹/⫹ mice
subjected to respective IR duration.
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Fig. 9. Renal expression of TGF-␤1 mRNA 24 h after IR for either 22.5 or 25
min in duration and after Sham in Smad3⫹/⫹ and Smad3⫺/⫺ mice. TGF-␤1
mRNA expression was determined by quantitative real-time RT-PCR and
standardized for 18S rRNA; n ⫽ 7– 8/group. *P ⬍ 0.05, Smad3⫺/⫺ vs.
Smad3⫹/⫹ mice subjected to respective IR duration.
TGF-␤1/Smad3 signaling (32). TGF-␤1 also engages Smad7,
which is a robust inducer of HO-1(16), a cytoprotective molecule that reduces acute kidney injury incurred by ischemia,
heme proteins, nephrotoxins, and endotoxins (19, 35, 43).
Presumably, the salutary effects of TGF-␤1 in ischemic renal
injury reflect the preferential elicitation or the preponderating
effects of cytoprotective rather than injurious signaling pathways. Our findings delineate that the effect of Smad3-dependent pathways is that of promoting, rather than protecting
against, acute ischemic renal injury.
A novel and important investigative line has recently demonstrated that prior exposure to the volatile anesthetic sevoflurane confers protection against acute ischemic renal injury
(26 –28). These studies demonstrate involvement of TGF-␤1
and Smad3 in mediating the protective effects of sevoflurane.
Included in these studies are control groups in which
Smad3⫺/⫺ mice were subjected to renal ischemia under pentobarbital sodium anesthesia; however, renal dysfunction, as
measured by serum creatinine, under these conditions was
comparable in Smad3⫹/⫹ and Smad3⫺/⫺ mice (26). In this
regard, we wish to point out the following considerations. First,
the model of ischemia used in these studies (unilateral ischemia
of 30 min and contralateral nephrectomy) differs from ours
(bilateral ischemia of a duration of 22.5 and 25 min), and this
difference in the model of renal ischemia as well as experimental conditions may be relevant. Second, the protective
effect we observed at a given duration of ischemia (22.5 min)
was reproduced at another duration (25 min). Third, we employed two different markers of glomerular filtration, namely,
BUN and serum creatinine. Fourth, in one of our protocols of
renal ischemia (22.5), the degree of renal dysfunction in
Smad3⫹/⫹ mice at 24 h (serum creatinine ⬃1.5 mg/dl) was
comparable to that observed in Smad3⫹/⫹ mice 24 h after
ischemia reported in prior studies.
In summary, to the best of our knowledge, we provide the
first demonstration that induction of Smad3 occurs following acute ischemic insults, and such induction of Smad3
represents a maladaptive response that promotes acute isch-
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Fig. 7. Renal expression of IL-6 mRNA 24 h after IR for either 22.5 or 25 min
in duration and after Sham in Smad3⫹/⫹ and Smad3⫺/⫺ mice. IL-6 mRNA
expression was determined by quantitative real-time RT-PCR and standardized
for 18S rRNA; n ⫽ 7– 8/group. *P ⬍ 0.05, Smad3⫺/⫺ vs. Smad3⫹/⫹ mice
subjected to respective IR duration.
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Smad3 DEFICIENCY PROTECTS AGAINST MURINE AKI
Table 1. mRNA expression assessed by quantitative real-time RT-PCR
Ischemia-Reperfusion
Sham
HO-1
MCP-1
TNF-␣
22.5 min
25 min
Smad3⫹/⫹
Smad3⫺/⫺
Smad3⫹/⫹
Smad3⫺/⫺
Smad3⫹/⫹
Smad3⫺/⫺
7.9 ⫾ 1.1
5.9 ⫾ 1.1
5.6 ⫾ 1.1
11.9 ⫾ 3.7
5.8 ⫾ 0.7
8.1 ⫾ 1.8
57.1 ⫾ 11.9
62.5 ⫾ 5.5
63.0 ⫾ 6.0
35.1 ⫾ 6.5
49.6 ⫾ 6.7
53.4 ⫾ 6.7
62.8 ⫾ 8.3
55.6 ⫾ 7.1
51.8 ⫾ 4.9
47.9 ⫾ 11.0
53.0 ⫾ 6.1
44.5 ⫾ 6.9
Values are means ⫾ SE and are the results of relative quantification performed against a standard curve constructed for each mRNA target, normalized for
expression of 18S rRNA and expressed in arbitrary units; n ⫽ 7– 8 for all groups. HO-1, heme oxygenase-1.
ACKNOWLEDGMENTS
We gratefully acknowledge the secretarial expertise of Tammy Engel.
GRANTS
These studies were supported by National Institutes of Health Grants
DK47060, HL55552, and HL85307 (K. A. Nath and J. P. Grande).
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the authors.
Fig. 10. Serum concentrations of IL-6 protein 24 h after IR for 25 min in
duration and after Sham in Smad3⫹/⫹ and Smad3⫺/⫺ mice. IL-6 serum levels
were determined by ELISA; n ⫽ 7– 8/group. *P ⬍ 0.05, Smad3⫺/⫺ vs.
Smad3⫹/⫹ mice subjected to IR.
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emic injury. We suggest that the adverse effects of Smad3
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other established mediators of chronic kidney disease [for
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23.
Smad3 DEFICIENCY PROTECTS AGAINST MURINE AKI