Post on 08-Apr-2017
LABORATORY STUDY
Effect of Nephrotoxins on Tubulointerstitial Injury and NF-kB Activation inAdriamycin Nephropathy
Gopala K. Rangan, Yiping Wang, Yuet-Ching Tay, Jason D. Coombes, and David C. H. HarrisCenter for Transplant and Renal Research, Westmead Millennium Institute, The University of Sydney, Westmead Hospital,
Westmead, Sydney, Australia
In a previous study we found that an episode of acute
subclinical nephrotoxicity with gentamicin (G) (but not that
induced by another proximal tubular cell nephrotoxin: ferric
nitrilotriacetate, FeNTA), paradoxically reduced the progression
of renal function and injury in uninephrectomized rats with
nephrotic glomerular disease due to Adriamycin nephropathy
(AN). Here, we hypothesized that subclinical exposure to G
reduces early renal cortical tubulointerstitial inflammation and
NF-kB activation in AN. To test this hypothesis, male Wistar
rats with established AN received either G (10, 40, or 80 mg/kg
by daily s.c.i. for 3 days), FeNTA (1.25, 5, or 10 mg/kg by a
single i.p.i.), or vehicle (n=8 per group), 13 to 15 days after
disease induction. Although G and FeNTA caused acute tubular
necrosis in a dose-dependant manner (day 17), only the highest
doses (10 mg/kg and 80 mg/kg) produced an acute elevation in
the serum creatinine. On day 33, chronic tubulointerstitial
inflammation (tubular atrophy, interstitial ED-1+/CD8+ cell
accumulation) and NF-kB activation were exacerbated only in
the groups that caused functional nephrotoxicity. These data
suggest that: 1) the protective effect of subclinical G
nephrotoxicity in chronic AN does not involve early changes
in interstitial inflammation or NF-kB activation; and 2) a single
episode of G exposure must be accompanied by clinically
apparent nephrotoxicity in order to accelerate progression in a
nonuremic model of chronic glomerular disease.
Keywords nephrotoxin, chronic tubulointerstitial inflammation,
glomerular disease
INTRODUCTION
Nephrotoxins such as aminoglycosides, radiocontrast,
amphotericin, and anaesthetic compounds cause acute re-
nal failure, in part, by direct injury to proximal tubular
epithelial cells (PTECs).[1] It widely accepted that expo-
sure to nephrotoxins during the course of chronic glo-
merular disease (CGD) could hasten progression to
end-stage kidney failure by exacerbating tubulointerstitial
injury.[2,3] Paradoxically, in a previous longterm study,
we found that an episode of subclinical nephrotoxicity
(defined as pathological renal injury without a rise in the
serum creatinine) induced by gentamicin [but not ferric
nitrilotriacete (FeNTA); another proximal tubular cell
nephrotoxin] reduced the decline in renal function,
glomerulosclerosis, and tubulointerstitial injury in a rat
model of CGD (Adriamycin nephropathy, AN).[4] The
mechanisms involved in this novel type of acquired resis-
tance were not known.
Nuclear factor (NF)-kB is a highly conserved family
of transcription factors that has a critical role in mediating
inflammation, apoptosis, and growth in chronic disease.[5]
In early AN, the renal cortical activation of NF-kB is in-
creased and correlated with the severity of tubulointerstitial
injury.[6] Moreover, suppression of NF-kB activation in
established AN, with the antioxidant, pyrrolidine dithio-
carbamate (PDTC), attenuated tubulointerstitial inflam-
mation.[6] These data suggest that NF-kB has an important
role in mediating disease progression in this model.
In other experimental systems (such as the repeated
exposure of monocytes to lipopolysaccharide) attenua-
tion of NF-kB has been postulated to be one of the
mechanisms involved in mediating acquired resistance to
further tissue injury.[7] These observations led us to
hypothesize that an episode of subclinical gentamicin
nephrotoxicity reduces renal cortical NF-kB activation in
AN. To investigate this hypothesis, the dose-dependant
Address correspondence to Dr. Gopala K. Rangan, Depart-
ment of Renal Medicine and Transplantation, Westmead
Hospital, Westmead, Sydney 2145, Australia; Fax: (61-2)
9633-9351; E-mail: g.rangan@wmi.usyd.edu.au
609
Renal Failure, 27:609–614, 2005
Copyright D 2005 Taylor & Francis Inc.
ISSN: 0886-022X print / 1525-6049 online
DOI: 10.1080/08860220500200437
Order reprints of this article at www.copyright.rightslink.com
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effects of a single episode of gentamicin- and FeNTA-
induced nephrotoxicity on renal cortical tubulointerstitial
injury and NF-kB activation in early AN were examined
in this study.
MATERIALS AND METHODS
Male Wistar rats (6–8 weeks old) (n=56) were
supplied by the Animal Care Facility, Westmead Hospital
and allowed free access to food and water. The AN was
induced on day 0, by a single intravenous injection of
doxorubicin hydrochloride (7.5 mg/kg).[6] On day 10,
baseline 24-hour urinary protein and creatinine were
measured. On day 13, rats were stratified into seven groups
(n=8 per group) according to body weight, baseline
proteinuria, and endogenous creatinine clearance (CrCl).
The groups received: 1) vehicle (saline, day 14 to day 16,
by s.c. or i.p.i.); 2) gentamicin (G) (10, 40, or 80 mg/kg,
day 14 to day 16, by s.c.i.) or FeNTA (1.25, 5, or 10 mg/kg,
by a single i.p.i. on day 16). Sterile FeNTA was prepared
as previously described[4] and gentamicin was obtained
from David Bull Laboratories (Melbourne, Australia). The
doses of nephrotoxins were determined from preliminary
experiments and a previous study,[4] and designed to
induce mild, moderate, or severe acute tubular necrosis in
AN. Higher doses of the nephrotoxins (>80 mg/kg
gentamicin or >10 mg/kg FeNTA) caused diffuse tubular
necrosis and early mortality due to severe acute renal
failure in AN, whereas doses less than 1.25 mg/kg FeNTA
or 10 mg/kg gentamicin caused no tubular necrosis.
Animals were pair fed, and body weight and food
intake were measured daily. Proteinuria and CrCl were
determined on days 17, 21, and 33. Groups of animals
were sacrificed either on day 17 (n=3 per group) or day
33 (n=5 per group) for analysis of acute and chronic
effects on tubulointerstitial injury, respectively. On the
day of sacrifice, animals were anesthetized with ketami-
ne:xylazine, a mid-line laparotomy was performed and
both kidneys were removed, as previously described.
Table 1
Serum creatinine (mmol/L) in the experimental groups
Time AN+vehicle
AN+gentamicin (mg/kg) AN+FeNTA (mg/kg)
10 40 80 1.25 5 10
Day 10 49±1 50±2 47±2 50±1 46±1 44±1 48±2
Day 17 58±3 56±3 81±22 253±33* 50±3 54±2 161±38*
Day 21 57±2 57±3 65±5 236±50* 54±2 56±2 66±4
Day 33 64±4 64±4 59±4 85±9 45±1 48±2 58±2
NB: Gentamicin and FeNTA were administered on days 14 to 16.*p<0.05 vs. AN+vehicle.
Figure 1. The acute effect of gentamicin (G, 10–80 mg/kg) and ferric nitrilotriacetate (Fe, 1.25–10 mg/kg) on cortical
tubulointerstitial injury in Adriamycin nephropathy (AN) on day 17. Areas of focal acute tubular necrosis are present, in a dose-
dependant manner, in the groups treated with nephrotoxins (Magnification, �400, PAS stain).
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The Animal Ethics Committee, University of Sydney at
Westmead Hospital, approved all experimental protocols.
Formalin-fixed paraffin sections (3 mm), were stained
with periodic-acid Schiff (PAS). Cortical tubulointerstitial
injury was assessed by both a semiquantitative scoring
system and quantitative morphometric analysis. For
semiquantitative analysis, 20 random cortical fields were
viewed at �200 magnification and graded according to the
percentage area occupied by tubular injury (tubular
atrophy, dilatation) and interstitial inflammation (0=nor-
mal; 1=less than 25%; 2=26%–50%; 3=51%–75%;
4=76%–100%). For quantitative morphometry, 10 ran-
dom cortical fields were viewed on a computer screen
using a video camera (�400). The cross-sectional cell
height of cortical tubules and interstitial volume, was
measured by line and area morphometric measurements
respectively, using image analysis software (Optimas
version 5.2, Optimas Corporation, Seattle, WA).[6] The
number of interstitial monocyte (ED-1) and cytotoxic
T-lymphocytes (OX-8) were assessed by immunohisto-
chemistry on acetone-fixed frozen sections. Nonspecific
staining was blocked with normal rabbit serum (1:5)
followed by either mouse anti-rat ED-1 (1:400, Serotec,
Oxford, England) or mouse anti-rat OX8 (1:100, Serotec,
Oxford, England) for 1 h at room temperature. Anti-mouse
rabbit antibody (1:50 in 1% rat serum, Dako Australia,
Sydney, Australia) and mouse peroxidase-antiperoxidase
complex (PAP, Z0259, 1:100; Dako Australia, Sydney,
Australia) were applied sequentially for 25 minutes each at
room temperature. The mean numbers of 3,3-diamino-
benzidine tetrahydrochloride positive ED-1 or OX-8 pos-
itive cells were determined from 10 nonoverlapping
cortical fields (�400, measuring 0.075 mm2 each).
Extraction of nuclear protein and electrophoretic
mobility shift assay for NF-kB and activator protein (AP)-
1 were performed as previously described.[6,8] Competi-
tion and supershift assay were performed to confirm the
specificity of the retarded bands.
All data were expressed as mean±SEM. Compar-
isons between experimental groups were performed using
Figure 2. The subacute effect of gentamicin (G) and ferric nitrilotriacetate (Fe) on tubulointerstitial inflammation in Adriamycin
nephropathy (AN) on day 33. Chronic tubulointerstitial inflammation in AN is exacerbated by high-dose G and Fe treatment (PAS
stain).
Figure 3. Semiquantitative cortical tubulointerstitial injury
score in vehicle (C)-, gentamicin- and ferric nitrilotriacetate
(FeNTA)-treated rats with Adriamycin nephropathy on day 33.
*P<0.05 vs. C.
611Nephrotoxins and Adriamycin Nephropathy
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the independent t-test and Mann-Whitney U test for
parametric and nonparametric data respectively. A P
value less than 0.05 indicated a significant difference
between groups.
RESULTS
No animals died during the study. Groups adminis-
tered the highest doses of FeNTA (10 mg/kg) and G (80
mg/kg), gained less body weight than vehicle (Day 33:
AN+vehicle: 295±18; AN+G: 244±13; AN+FeNTA:
272±15 g; P=0.05). Body weight in the other groups was
similar to vehicle-treated animals. Serum creatinine was
elevated only in groups that received the highest doses of
FeNTA and G (Table 1). The recovery of the serum
creatinine occurred earlier in the FeNTA group (day 22),
whereas it was only partial by day 33 in the G group
(Table 1). The serum creatinine (or CrCl, data not shown),
did not change significantly in the other groups receiving
nephrotoxins (compared to vehicle) (Table 1), even
though acute tubular necrosis did occur at these lower
doses in AN (see the following information). There were
no differences in urinary protein excretion in the experi-
mental groups at any timepoint (urinary protein:creatinine
ratio on day 33: AN+vehicle: 72.2±6.5; AN+G: 76.4±
8.1; AN+FeNTA: 77.8±5.1 mg/mmol; P=not significant).
Histological examination on day 17 confirmed that
animals treated with either nephrotoxin developed dose-
dependant degrees of acute tubular necrosis (Figure 1).
On day 33 the histological changes of AN consisted of
cortical tubular atrophy, interstitial cell accumulation, and
Table 2
Quantitative morphometric analysis of the tubulointerstitial inflammation in the experimental groups on day 33
Group n
Dose
mg/kg
Interstitial
volume %
Mean tubule
cell height (mm)
Interstitial
ED-1 cells/hpf
Interstitial
CD8 cells/hpf
AN+vehicle 5 — 12.2±1.6 14.9±3.0 6.64±0.68 8.16±1.30
AN+gentamicin 5 80 33.0±6.5* 8.6±0.6* 35.78±3.56* 11.90±0.70*
AN+FeNTA 5 10 26.9±5.8* 9.4±0.3* 18.92±3.32* 11.58±2.45*
*p<0.05 vs. AN+vehicle.
Figure 4. The DNA-binding activity of nuclear factor (NF)-kB (A) and activator protein (AP)-1 (B) in renal cortical nuclear
extracts from vehicle (C)-, gentamicin-, and ferric nitrilotriacetate (FeNTA)-treated rats with Adriamycin nephropathy on day 33 of
the study. Top and lower panels show representative autoradiographs and mean densitometry (n=5 per group) respectively. *P<0.05
vs. C.
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volume expansion (Figure 2). By semiquantitative anal-
ysis, cortical tubulointerstitial injury was exacerbated
only in the groups that received the highest doses of either
FeNTA or G (Figures 2 and 3). By quantitative mor-
phometric analysis, interstitial volume and cortical
tubular atrophy (as assessed by a reduction in cross-
sectional tubule cell height) were increased in these
groups, compared to vehicle (Table 2). Similarly,
interstitial ED-1 and CD8 cell accumulation in AN was
worsened by high-dose FeNTA and G (Table 2).
The DNA-binding activity of both AP-1 and NF-kB
were increased in renal cortical nuclear extracts from rats
with AN. On day 33, NF-kB DNA-binding activity (but
not AP-1) was exacerbated only in the groups that
received the highest doses of either FeNTA or G,
compared to vehicle (Figure 4).
DISCUSSION
The results of this study demonstrate that the acute
administration of nephrotoxic doses of two site-specific
proximal tubular cell nephrotoxins (gentamicin and
FeNTA) to rats with established AN exacerbated under-
lying tubulointerstitial inflammation and renal cortical
NF-kB activation. In contrast, lower doses, that caused
mild acute tubular necrosis and subclinical nephrotoxicity,
were not sufficient to alter early tubulointerstitial inflam-
mation or renal cortical NF-kB activation.
Previously, in a uninephrectomized model of Adria-
mycin nephropathy, we reported that an episode of
subclinical nephrotoxicity induced by gentamicin (but
not FeNTA) at week 6, completely prevented the decline
in creatinine clearance and partially reduced glomerulo-
sclerosis and tubulointerstitial fibrosis at week 14.[4] The
present study was performed to investigate the potential
role of NF-kB in mediating the protective effect of
gentamicin in AN. However, we were unable to demon-
strate that an early downregulation in NF-kB activation (as
described in some models of acquired resistance)[7] and
tubulointerstitial inflammation were mechanisms to ex-
plain the protective effect of subclinical gentamicin
nephrotoxicity on the longterm progression of chronic
AN. Other possibilities that could be involved include the
upregulation of antioxidant proteins (such as heme-
oxygenase-1)[9] and/or specific effects of gentamicin on
the lysosomal processing of filtered plasma proteins.[4]
In previous studies, inhibition of renal cortical NF-
kB activation with PDTC reduced cortical tubulointer-
stitial injury in AN.[6] In the present study, exacerbation
of interstitial inflammation and tubular atrophy with
nephrotoxins was associated with augmentation of renal
cortical NF-kB (but not AP-1). These correlative data
provide further evidence that NF-kB activation is
involved in the pathogenesis of chronic tubulointerstitial
injury in progressive renal diseases.
Avoiding exposure to nephrotoxins is one of the main
therapeutic cornerstones in the clinical management of
preventing disease progression in chronic kidney disease.
Our experimental data suggests that an episode of
subclinical nephrotoxicity, at least following a short
exposure to gentamicin, does not have any adverse long-
term effects in a nonuremic nephrotic model of glomerular
disease. However, the clinical scenario in humans is more
complex than that that can be replicated in the laboratory.
In the clinical setting, patients have impaired renal
function and are exposed to multiple nephrotoxic insults,
sometimes on repeated occasions. This may be an
explanation for the divergence of our experimental data
with retrospective clinical studies. For example, Aubia and
colleagues showed that subclinical nephrotoxin exposure
(including aminoglycosides and possibly anesthesia)
accelerated the decline in renal function by more than 2-
fold in humans with impaired renal function due to
diabetic nephropathy.[10] Similarly, aminoglycoside expo-
sure accelerated the decline in loss of residual renal
function in peritoneal dialysis patients with end-stage
renal failure.[11,12]
In summary, the cytoprotective effect of subclinical
gentamicin nephrotoxicity in AN is not due to an early
downregulation in renal cortical NF-kB activation, and
involves other mechanisms that require further investiga-
tion. In addition, the acute exposure to clinically
nephrotoxic doses of gentamicin and FeNTA worsened
renal cortical tubulointerstitial injury in AN through
mechanisms that may involve the upregulation of NF-kB.
ACKNOWLEDGMENTS
This study was supported by the Australian Kidney
Foundation (GKR) and project grant 970721 from the
National Health Medical Research Council of Austra-
lia (DCH).
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