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Dietary sodium restriction prevents kidney damage
in high fructose-fed rats
Carole Oudot
1
, Anne D. Lajoix
1
, Bernard Jover
1
and Caroline Rugale
1
1
Groupe Rein et Hypertension, FRE3400 CNRS/University, Montpellier, France
Sodium depletion has a protective effect on target-organ
damage in hypertension independent of blood pressure.
Here we tested whether chronic dietary sodium restriction
may prevent the development of renal alterations associated
with insulin resistance by reducing the inflammatory and
oxidant state. Rats were fed normal-salt–60% fructose, low-
salt–60% fructose, or control normal-salt diet for 12 weeks.
Insulin resistance induced by high-fructose diet was
associated with an increase in albuminuria, tubular and
glomerular hypertrophy, and inflammation of kidney and
adipose tissue. The low-salt diet improved insulin sensitivity
and prevented kidney damage. These beneficial effects of
sodium depletion were associated with a decrease in renal
inflammation (macrophage infiltration, IL-6, TNF-a)and
oxidative stress (NADPH oxidase activity), and a prevention of
histologic changes in retroperitoneal fat induced by high
fructose. Thus, dietary salt depletion has beneficial effects on
renal and metabolic alterations associated with a high-
fructose diet in rats.
Kidney International (2013) 83, 674–683; doi:10.1038/ki.2012.478;
published online 23 January 2013
KEYWORDS: albuminuria; dietary sodium; IL-6; insulin resistance; oxidative
stress; TNF-a
The metabolic syndrome was first introduced as a concept to
cluster cardiovascular risk factors such as hypertension, type
2 diabetes, obesity, and dyslipidemia, which culminate in
high risk for atherosclerotic cardiovascular disease. Insulin
resistance has been proposed as the metabolic link between
all these cardiovascular risk factors.1–3 Recently, consumption
of dietary fructose was suggested to be one of the
environmental factors contributing to the development of
obesity and the accompanying abnormalities of the metabolic
syndrome.4In fact, the high consumption of fructose is a
well-known experimental model of metabolic syndrome.5,6
Several studies have suggested that renal damages are
associated with the metabolic syndrome. In humans, the
multivariate-adjusted odds ratio of chronic kidney disease
and microalbuminuria were higher in patients with the
metabolic syndrome.7Similarly, it has been shown that
fructose-fed rats in comparison with corn starch–fed rats
show signs of renal dysfunction associated with renal
hypertrophy, afferent arteriolopathy, glomerular hyperten-
sion, renal vasoconstriction, and nephropathic changes.8In
addition, long-term (46 months) exposure to high fructose
consumption was associated with proteinuria, kidney
enlargement, and focal tubulointerstitial injury and
glomerulosclerosis.9,10 Accumulating evidence now indicates
that immunological and inflammatory mechanisms have a
significant role in the development and progression of
damages in chronic kidney diseases. Among the various
inflammatory cytokines, mainly tumor necrosis factor-a
(TNF-a) and interleukin-6 (IL-6) are relevant mediators that
regulate inflammatory immune responses in diabetic
nephropathy.11 Interestingly, there is increasing body of
evidence suggesting that insulin resistance may result from an
inflammatory disorder, in which macrophages in adipose
tissue and elsewhere may have an important role.12 Together
with inflammation, oxidative stress seems to be implicated in
renal injury in obesity and hypertension13, as well as in type 2
diabetic nephropathy in mice and in fructose-fed rats.5,14
Sugar consumption has also been linked to sodium intake
and renal sodium handling. Fructose–blood pressure associa-
tion was stronger for individuals with high sodium
consumption,15 as well as in spontaneously hypertensive
rats fed a high-sodium diet.16 Conversely, a low-sodium diet
prevented changes in blood pressure induced by high dietary
sucrose17 or fructose.16 Besides its effect on arterial pressure,
basic research http://www.kidney-international.org
&2013 International Society of Nephrology
Correspondence: Caroline Rugale, Groupe Rein et Hypertension, CNRS
FRE3400, 641 Avenue du Doyen GIRAUD, 34093 Montpellier Cedex 5, France.
E-mail: caroline.rugale@inserm.fr
Received 20 June 2012; revised 18 October 2012; accepted 8 November
2012; published online 23 January 2013
674 Kidney International (2013) 83,674–683
we have previously reported that sodium withdrawal from
the diet alleviated cardiovascular and renal damages in
angiotensin II hypertension. The beneficial influence of
dietary sodium restriction was independent of arterial
pressure reduction and possibly related to attenuation of
the prooxidant effect of the peptide.18 However, the effects of
dietary sodium restriction on functional and morphological
modifications of the kidney associated with insulin resistance
have not been yet explored. Thus, in the present study, we
evaluated the putative beneficial influence of a drastic
reduction in sodium intake on fructose-induced renal
alterations. Furthermore, we tested the hypothesis that
sodium restriction was related to a reduction in the
inflammatory and oxidative stress responses to high
fructose–induced insulin resistance in rats. As the podocyte
is a key cell type involved in the initial development of
albuminuria, particularly in obesity or type 2 diabetes,19
renal ultrastructure was examined in fructose-fed rats.
Finally, we evaluated the influence of sodium restriction on
retroperitoneal adipose tissue, which may have a role in the
development of insulin resistance.20
RESULTS
Metabolic parameters
Despite a higher calorie intake in fructose-fed rats, the final
body weight was similar in sodium-replete, fructose-fed
(normal-sodium, high-fructose (NSF)) and control group
(normal-sodium control (NSC)) rats. However, the percen-
tage of visceral adipose tissue was significantly higher in NSF
rats (Table 1). Interestingly, body weight was lower in low-
sodium, fructose-fed rats (low-sodium, high-fructose (LSF)),
and sodium restriction prevented the increase in adipose
tissue associated with the fructose diet. As reported in
Table 1, plasma concentration of uric acid was higher in the
NSF group as compared with the control group, and sodium
depletion had no effect on this change. Plasma adiponectin
was significantly decreased in NSF rats as compared with
NSC rats, and sodium restriction did not influence this
change. Plasma lectin concentration was similar in all groups.
No significant influence of high fructose intake on sodium
and potassium excretion was observed. As expected, sodium
excretion was reduced to o50 mmol per day in the LS-fed
rats. Direct and indirect blood pressure was comparable
among the three experimental groups.
Glucose metabolism
Although fasting plasma glucose was not significantly
different between groups, fasting plasma insulin and home-
ostasis model assessment (HOMA) index increased in NSF
rats by 59% and 78%, respectively (Table 1). In addition,
blood glucose response during insulin tolerance test was
blunted in the NSF group when compared with the NSC
group (Figure 1a). Sodium restriction precluded insulin
resistance as evaluated by both HOMA index and insulin
tolerance test.
As depicted in Figure 1b, blood glucose response to
intraperitoneal glucose tolerance test was comparable in the
three groups. However, insulin response was enhanced in the
NSF rats, and significance was achieved when the area under
the curve was calculated. Both the peak and area under the
curve of plasma insulin in response to intraperitoneal glucose
tolerance test were similar to that of NSC rats in animals fed
the sodium-depleted fructose diet.
Renal morphology and histology
No significant difference in inulin clearance and plasma
creatinine was observed among the three groups (Table 2).
As depicted in Figure 2, a high-fructose diet was associated
with a marked rise in albuminuria in sodium-replete
rats. Albuminuria was comparable to standard control rats
Table 1 | Influence of high-fructose diet and sodium depletion on experimental parameters (N¼8 in each diet)
NSC NSF LSF
Final body weight, g 453±8434
±10 379±13*
,w
Food intake, g/day 19±219
±120
±1
Calories intake, kcal/day 56±676
±4* 76±7*
Adipose tissue, % BW 1.84±0.13 2.47±0.17* 1.68±0.18w
Sodium excretion, mmol/day 1.64±0.08 1.58±0.16 0.03±0.00*
,w
Potassium excretion, mmol/day 1.70±0.17 1.56±0.09 1.93±0.13
Urine volume, ml/day 12±113
±29
±1
Plasma glucose, mg/dl 126±10 121±3104
±5
Plasma insulin, ng/ml 1.19±0.10 1.89±0.29* 1.44±0.18*
HOMA-IR 1.58±0.15 2.81±0.44* 1.82±0.21w
Plasma uric acid, mg/dl 1.64±0.21 2.59±0.35* 2.20±0.29
Plasma adiponectin, mg/ml 22.8±3.8 14.8±0.6* 11.9±1.7*
Plasma leptin, ng/ml 5.6±1.3 5.1±0.7 6.1±0.9
Tail-cuff pressure, mmHg 136±3137
±3136
±3
Vigil mean arterial pressure, mm Hg 121±4122
±4123
±5
Abbreviations: BW, body weight; HOMA-IR, homeostasis model assessment of insulin resistance; LSF, low-sodium, high-fructose diet; NSC, normal-sodium control diet; NSF,
normal-sodium, high-fructose diet.
*Po0.05 versus NSC diet.
w
Po0.05 versus NSF diet.
Food and calories intake, and urinary excretion of sodium, potassium, and volume are expressed as a daily mean value calculated over a 3-day collection period.
C Oudot et al.: Sodium, insulin resistance, and kidney basic research
Kidney International (2013) 83, 674–683 675
in sodium-deprived fructose-fed rats. Renal mass was
significantly higher in NSF rats as compared with NSC rats
(Table 2). Kidney hypertrophy induced by fructose diet
was associated with an increase in proximal tubular
and glomerular area. The glomerulomegaly associated with
the high-fructose diet was completely prevented and the
tubulopathy was reduced in rats fed the low-sodium, fructose-
enriched diet.
Sirius red staining of cortical tissue showed a significant
increase in NSF but not in LSF rats, thus indicating a higher
glomerular and interstitial collagen deposition in the former
group (Figure 3).
Time after insulin injection (min)
Blood glucose (% of basal blood glucose)
20
40
60
80
100
0 1530456075
NSC
NSF
LSF
LSFNSFNSC
0
2000
4000
6000
Glucose AUC (mg/dl/min)
*
†
Glucose AUC (mg/min/dl)
Time after glucose injection, min
0
100
200
300
400
NSF
LSF
NSC
0.00
4.00
8.00
12.00
16.00
Blood glucose (mg/dl)
Plasma insulin (ng/ml)
0 15306090
Insulin AUC (ng/min/ml)
0
5000
10,000
15,000
20,000
NSFNSC
0
100
200
300
400
500 *
LSF
†
Figure 1 | Insulin and glucose tolerance tests in rats fed a high-fructose diet with normal or low sodium content. (a) Insulin tolerance
test (ITT). (b) Intraperitoneal glucose tolerance test (IPGTT). N¼8 in each diet. Data are shown as means±s.e.m. *Po0.05 versus
NSC diet;
w
Po0.05 versus NSF diet. AUC, area under the curve; LSF, low-sodium, high-fructose diet; NSC, normal-sodium control diet;
NSF, normal-sodium, high-fructose diet.
Table 2 | Influence of high-fructose diet and sodium depletion on kidney function and morphology (N¼8 in each diet)
NSC NSF LSF
Plasma creatinine, mg/dl 0.41±0.05 0.53±0.06 0.50±0.04
Inulin clearance, ml/min per g KW 1.02±0.09 0.96±0.07 0.82±0.09
Kidney weight, g 2.62±0.08 3.08±0.13* 2.79±0.11
Proximal tubular area, mm
2
1670±135 2523±269* 1844±197
w
Nucleus/epithelial area, 10
3
nucleus/mm
2
5.9±0.3 6.6±0.7 5.3±0.3
Epithelial area/nucleus, mm
2
/nucleus 180±10 200±27 192±12
Glomerular area, mm
2
13,676±830 16,012±801* 13,271±510
w
Bowman’s space area, mm
2
4918±364 6249±410* 4642±253
w
Abbreviations: KW, kidney weight; LSF, low-sodium, high-fructose diet; NSC, normal-sodium control diet; NSF, normal-sodium, high-fructose diet.
*Po0.05 versus NSC diet.
w
Po0.05 versus NSF diet.
676 Kidney International (2013) 83,674–683
basic research C Oudot et al.: Sodium, insulin resistance, and kidney
Similarly, enhanced macrophage infiltration, as evidenced
by infiltration of ED1-positive cells, was also observed in NSF
animals compared with NSC rats. Interestingly, macrophage
infiltration was normalized in LSF rats (Figure 3).
Renal levels of IL-6 and TNF-awere twofold higher in
fructose-fed rats with a normal salt content as compared
with rats fed the standard chow. Dietary sodium depletion
blunted the increase in both inflammation mediators
(Figure 4a).
NADPH oxidase activity in the kidney
High-fructose diet was associated with a marked increase in
the velocity of nicotinamide adenine dinucleotide phosphate
(NADPH) oxidase activity and the cumulative superoxide
anion (O2
1
) production in the kidney, which were both
prevented by LS diet (Figure 4b).
Filtration barrier integrity evaluation by glomerular desmin
immunostaining and ultrastructural analysis
Immunostaining for desmin was increased in the glomeruli
of NSF rats compared with the control and was in turn
reduced in LSF rats (Figure 5a and b).
As depicted in Figure 6a, the foot process fusion and
effacement observed in NSF rats was improved in low-
sodium, fructose-fed rats. Quantitative analysis showed that
sodium restriction was associated with an increase in the slit
pore diaphragm diameter and a reduction of podocyte base
width (Figure 6b).
Adipose tissue histology
As reported in Figure 7, adipocyte size of retroperitoneal
fat was higher in NSF rats as compared with NSC rats.
In contrast, adipocyte size of epididymal adipose tissue
was similar in fructose-fed rats compared with rats on the
control diet (2991±148 and 2695±92 mm
2
in NSF and
NSC, respectively). In addition, macrophage infiltration in
retroperitoneal adipose tissue was greater in high fructose–fed
rats. Sodium restriction prevented the increase detected on
either retroperitoneal or epididymal adipose tissue changes
Urinary albumin excretion (μg/24 h)
NSC NSF LSF
1
10
100
1000
10,000
*†
Figure 2 | Influence of high-fructose diet and sodium depletion
on urinary albumin excretion. N¼8 in each diet. Data are shown as
means±s.e.m. *Po0.05 versus NSC diet;
w
Po0.05 versus NSF diet.
LSF, low-sodium, high-fructose diet; NSC, normal-sodium control diet;
NSF, normal-sodium, high-fructose diet.
NSC NSF LSF
50 μm
50 μm
3
2
1
0
NSC NSF LSF
*
60
40
20
0NSC NSF LSF
*
Collagen stalning
(%)
ED1 cells/mm2
†
†
Figure 3 | Fibrosis and macrophage infiltration in the kidney. (a) Representative microphotographs of Sirius red staining (top panel) and
immunohistochemistry for ED1 (bottom panel) in the cortex. Arrows indicate ED1-positive macrophages. Scale bar ¼50 mm. (b) Quantitative
analysis of collagen deposition (% stained area in glomeruli and tubulointerstitium) and ED1-positive macrophage infiltration (number of
positive cells per mm
2
of tissue). N¼8 in each diet. Data are shown as means±s.e.m. *Po0.05 versus NSC diet;
w
Po0.05 versus NSF diet.
LSF, low-sodium, high-fructose diet; NSC, normal-sodium control diet; NSF, normal-sodium, high-fructose diet.
Kidney International (2013) 83, 674–683 677
C Oudot et al.: Sodium, insulin resistance, and kidney basic research
associated with fructose consumption. These retroperitoneal
adipose tissue changes associated with fructose diet were not
observed with sodium depletion.
DISCUSSION
As expected, rats fed a high-fructose diet for 12 weeks
developed insulin resistance, characterized by hyperinsuline-
mia without hyperglycemia and alterations of the response to
exogenous glucose and insulin administration. Insulin
resistance was associated with a higher urinary albumin
excretion, an early predictor of diabetic nephropathy, and
premature cardiovascular disease.21 The lack of detectable
changes in glomerular filtration rate and in arterial pressure
does not favor a major implication of hemodynamic changes
in the enhanced albuminuria in this model. Surprisingly, no
significant increase in blood pressure was recorded in the
present sodium-replete, fructose-fed rats. Of note, the
absolute level of blood pressure achieved in the fructose-fed
rats (135–140 mm Hg) was similar to that reported by
others.6,8 Discrepancies may therefore be more related to
the blood pressure level of control rats, which was currently
higher than in other reports (136 vs. 105–110 mm Hg).
Interestingly, blood pressure was not affected by dietary
sodium restriction, thus suggesting that its beneficial renal
influence was unlikely to be related to systemic hemodynamic
effect in the present model.
Among the kidney changes observed in the present study
are increases in renal mass, proximal tubular and glomerular
hypertrophy, and interstitial fibrosis. Although our study was
not designed to address this question, an increased sensitivity
LSFNSFNSC
0
2
4
6
8
0
2000
4000
6000
8000
10,000
LSFNSFNSC
NADPH oxidase velocity (RLU/s) O2
°– production (RLU per mg tissue)
**
††
0
4
8
12
16
20
LSFNSFNSC
*
†
Kidney TNFα (pg per mg tissue)
LSFNSFNSC
†
*
Kidney IL-6 (pg per mg tissue)
0
0.5
1
1.5
2
2.5
3
3.5
Figure 4 | Inflammation and oxidative stress in the kidney. (a) Interleukin-6 (IL-6) and tumor necrosis factor-a(TNF-a) level in kidney
homogenate. (b) Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase velocity and cumulative superoxide anion (O2
1
) production
in kidney homogenate. N¼8 in each diet. Data are shown as means±s.e.m. *Po0.05 versus NSC diet;
w
Po0.05 versus NSF diet. LSF, low-
sodium, high-fructose diet; NSC, normal-sodium control diet; NSF, normal-sodium, high-fructose diet; RLU, relative light units.
NSC NSF LSF 4
3
2
1
0NSC NSF LSF
Desmin stalning
(% glomerular tuft)
*
†
Figure 5 | Glomerular desmin immunostaining. (a) Representative microphotographs of glomerular desmin immunostaining. Bar ¼50 mm.
(b) Quantitative analysis of desmin immunostaining expressed in percent of glomerular tuft. Data are shown as means±s.e.m. N¼5 in each
diet. *Po0.05 versus NSC diet;
w
Po0.05 versus NSF diet. LSF, low-sodium, high-fructose diet; NSC, normal-sodium control diet; NSF, normal-
sodium, high-fructose diet.
678 Kidney International (2013) 83,674–683
basic research C Oudot et al.: Sodium, insulin resistance, and kidney
to sodium, even at a regular dosage, may participate in the
renal damages of fructose-fed rats. Yet, similar kidney
changes associated with a high-fructose diet have been
previously reported. Renal hypertrophy with tubular cell
proliferation and low-grade tubulointerstitial injury was
observed after 6 weeks of high-fructose diet.22 A glomerular
hypertrophy also occurred both at an early state (4 weeks)
and after a long-term high-fructose diet.23 In addition,
NSC NSF LSF
350
a
b
300
250
200
150
100
50
0NSC NSF LSF
*80
60
40
20
0NSC NSF LSF
*
Slit pore
diameter (nm)
Podocyte foot process
base width (nm)
†
†
Figure 6 | Ultrastructural depiction of podocyte foot processes in NSF and LSF rats with transmission electron microscopy. (a) Center
panel depicts podocyte foot process effacement (black arrows) with increases in podocyte base width and loss of the slit pore diaphragm in
NSF rats. Right panel depicts the uniformity of podocytes processes in LSF rats. (b) Ultrastructural analysis of glomerular filtration barrier
measures. N¼5 in each diet. Data are shown as means±s.e.m. *Po0.05 versus NSC diet;
w
Po0.05 versus NSF diet. Bar ¼500 nm. LSF,
low-sodium, high-fructose diet; NSC, normal-sodium control diet; NSF, normal-sodium, high-fructose diet.
NSC NSF LSF
7000
50 μm
6000
5000
4000
3000
2000
1000
0
NSC NSF LSF
*
Adipocyte area (μm2)
60
50
40
30
20
10
0
ED1 cells/mm2
*
NSC NSF LSF
†
†
Figure 7 | Adipocyte size and macrophage infiltration in retroperitoneal adipose tissue. (a) Representative microphotographs of
hematoxylin–eosin staining and immunohistochemistry for ED1 in adipose tissue. Bar ¼50 mm. (b) Quantitative analysis of adipocyte area (mm
2
)
and ED1-positive macrophage infiltration (number of positive cells per mm
2
of adipose tissue). Data are shown as means±s.e.m. N¼8 in each
diet. *Po0.05 versus NSC diet;
w
Po0.05 versus NSF diet. LSF, low-sodium, high-fructose diet; NSC, normal-sodium control diet; NSF, normal-
sodium, high-fructose diet.
Kidney International (2013) 83, 674–683 679
C Oudot et al.: Sodium, insulin resistance, and kidney basic research
micropuncture studies documented that fructose intake
results in glomerular hypertension and reduced renal blood
flow in association with the development of preglomerular
vascular disease.24
Various cytokines are involved in the initial stage of
diabetic nephropathy and may represent renal markers of
early target-organ damage.11 In the current fructose-fed rats,
renal damages were associated with macrophage infiltration,
and increased TNF-aand IL-6 levels. In addition, renal
NADPH oxidase activity and O2
1
production were
augmented in sodium-replete, fructose-fed rats. Previous
studies have demonstrated a significant role of TNF-ain the
development of renal hypertrophy and induction of reactive
oxygen species in mesangial cells.25 TNF-ais also known to
stimulate IL-6 production by mesangial cells in a paracrine
manner.26 IL-6 mediates renal injury by alteration in
endothelial permeability, induction of mesangial cell
proliferation, and increased fibronectin expression.27 Recent
experimental studies show that excretion of TNF-aduring
diabetes was directly associated with a reduction in renal
sodium retention, renal hypertrophy, and urinary albumin
excretion.28 Moreover, TNF-aenhances the local generation
of reactive oxygen species, enhancing albumin permeability
through alteration of the glomerular capillary wall barrier.29
Altogether, these findings favor a direct association between
TNF-aand renal damage, particularly albuminuria in insulin
resistance and established diabetic nephropathy.
A major finding of the present work is the prevention by
dietary sodium restriction of the increase in albuminuria, as
well as all the observed kidney changes associated with
fructose-induced insulin resistance. We have previously
reported a beneficial influence of sodium restriction on
albuminuria in hypertensive rats,18 and such an effect was
also observed on blood pressure in fructose-fed rats.30 In the
kidney of the current fructose-fed rats as observed in the
cardiovascular system,18 dietary sodium restriction was
accompanied by a reduction in O2
1
production.
Moreover, the renal beneficial effect of the sodium-deprived
diet was associated with an anti-inflammatory effect equated
with the decrease in macrophage infiltration and the lower
cytokine production in the renal tissue. Among the possible
mechanisms that may be involved in the influence of the low-
sodium diet, a blunting of the oxidative and inflammatory
effects of angiotensin II might occur in this model.
Angiotensin II is known to be an important contributor of
the development of insulin resistance31,32, and it induces
oxidative stress, nuclear factor-kB activation, and increased
TNF-aas documented in skeletal muscle from insulin-
resistant Ren-2 rats.33 Recently, a role of IL-6 in angiotensin
II–mediated hypertension was demonstrated in humans.
Although IL-6 and C-reactive protein levels increased after
infusion of angiotensin II in normotensive and hypertensive
subjects, none of these inflammatory markers were elevated
with the activation of the endogenous renin–angiotensin
system by a low-salt diet period of 7 days.34 The findings
obtained in humans and those presented in our rat model
suggest that the potential protective effect of very
low–sodium diet results from a complex modulation of the
renin–angiotensin system. Although oxidative stress and
inflammation seem to be involved, the precise mechanisms
of the beneficial influence of low sodium consumption have
to be further investigated.
Previous work related to the pathogenesis of albuminuria
in type 2 diabetes has delineated abnormalities of the
filtration barrier.35 The influence of the low-sodium diet on
filtration barrier integrity was evaluated in our model of
insulin resistance by determining desmin immunostaining
and glomerular ultrastructure. The increase in podocyte
injury observed in NSF-fed rats was very likely to be due to
the subsequent modifications associated with fructose diet
rather than a direct effect of the sugar. Interestingly, podocyte
damage was prevented by sodium restriction. In addition,
electron microscopy measurements demonstrated that
podocyte foot process effacement, loss of slit pore
diaphragm integrity, and widening of the bases of the
podocyte foot process observed in rats fed a high-fructose
diet were improved when sodium was withdrawn from the
fructose diet. An association between insulin resistance,
oxidative stress, and glomerular filtration barrier injury was
previously reported in Zucker obese rats.36 This relation was
supported by accumulating data in favor of a susceptibility of
the podocyte to immunologic and inflammatory injury from
reactive oxygen species generated by endothelial cells.35,37
Interestingly, both abnormalities, that is, increased reactive
oxygen species generation and loss of glomerular filtration
integrity, were prevented by sodium restriction in the current
work, thus suggesting that oxidative stress is an important
factor of the beneficial influence of sodium depletion on
renal changes associated with a high-fructose diet.
Adipocyte and adipose tissue dysfunction are primary
defects in obesity and may link obesity to insulin
resistance and increased risk of type 2 diabetes, hyper-
tension, dyslipidemia, and atherosclerosis.38,39 Adipocyte
dysfunction was also associated with a downregulation of
adiponectin, an anti-inflammatory adipokine.40 In the
current experiments, high fructose–fed rats developed more
adiposity and had greater-sized adipocytes in retroperitoneal
adipose tissue as compared with rats fed standard chow, and
was associated with an increase in macrophage infiltration
and a decrease in plasma adiponectin. As previously
reported,41 plasma leptin level was unchanged after 12
weeks of fructose diet. Interestingly, sodium restriction
prevented both the increase in adipocyte size and
macrophage infiltration in retroperitoneal fat without
change in plasma adiponectin and leptin level as compared
with normal-salt, fructose-fed rats. This suggests that the
beneficial effect of the low-salt diet on inflammation was not
mediated by these two adipokines. Macrophage infiltration
into fat was shown to be positively correlated with adipocyte
hypertrophy, and to be a source of inflammatory
cytokines,42,43 leading to the development of a low-grade
chronic inflammatory state. Macrophages also have a
680 Kidney International (2013) 83,674–683
basic research C Oudot et al.: Sodium, insulin resistance, and kidney
reciprocal relationship with adipocytes. Fatty acids released
by adipocytes stimulate TNF-arelease by macrophages,
which, in turn, can stimulate the production of IL-6 by fat
cells, further amplifying the inflammatory response in
adipose tissue, as well as the kidney.44 Then, we can
hypothesize that the beneficial effect of dietary sodium
restriction on the inflammation of adipose tissue in the
current work could participate in its preventive effect on
kidney damage.
The glucose metabolism change is another factor that
could play a role in the beneficial renal effect of the low-salt
diet in the present study. Indeed, mounting evidence
supports a role for hyperinsulinemia in glomerular filtration
barrier injury and subsequent albuminuria observed in type 2
diabetes mellitus.35,45 The present results indicate that low
salt intake prevents insulin resistance and hyperinsulinemia
induced by long-term high-fructose diet.
Low sodium intake was associated with a reduced body
growth, which may be related to sodium and water losses
during the early phase of adaptation18 as well as a direct effect
on adipose tissue. One cannot exclude that low sodium
intake partly prevented insulin resistance through reduction
of body growth, particularly in adipose tissue. Dietary
sodium restriction may also improve insulin resistance
through the prevention of a rise in plasma uric acid. Uric
acid was proposed to mediate insulin resistance in fructose-
fed rats46 even if plasma concentration was not increased in
an earlier stage (5 weeks).47 In the present rats fed for 12
weeks with fructose, plasma uric acid was increased when
compared with control rats. Yet, sodium restriction did not
modify the elevated level of uric acid. Changes in uric acid
are therefore unlikely to be involved in the improvement of
insulin sensitivity observed in sodium-restricted, fructose-fed
rats.
Contrasting findings have been reported on the effect of
salt restriction on insulin sensitivity in normal rats. Most of
the studies48–50 showed that salt restriction lowers insulin
sensitivity, although some studies concluded the opposite.51
The decrease in insulin sensitivity observed in rats fed a low-
sodium diet from weaning to adulthood was not associated
with renin–angiotensin activity or body mass changes.50
However, all studies from the literature were conducted on
standard rats or healthy subjects with basal normal insulin
sensitivity. To our knowledge, this is the first study evaluating
the impact of salt restriction in a model of insulin resistance
in the rat and comparing it with a normal-salt diet. The
decrease in adiposity and adipocyte size and the reduction of
macrophage infiltration in visceral adipose tissue could partly
explain the beneficial effect of sodium restriction on glucose
metabolism.
To conclude, the present results indicate that dietary
sodium restriction has a beneficial influence on renal and
metabolic alterations associated with a high-fructose diet. It
is suggested that the renal reduction in oxidative stress and
proinflammatory cytokines is very likely to be related to the
prevention of subsequent podocyte injury and fibrosis. A
possible link between adipose tissue and renal damages as
suggested in childhood–adolescent obesity52 deserves further
investigation in the beneficial influence of dietary sodium
restriction in fructose-fed rats.
MATERIALS AND METHODS
The present animal experiments complied with the European and
French laws (permit numbers B-3417226 and 34179) and conform
to the Guide for the Care and Use of Laboratory Animals published
by the National Institutes of Health (publication no. 85-23, revised
1996). Rats were housed in climate-controlled conditions with a 12-
h light/dark cycle in a temperature-controlled room (22±11C).
Animals and metabolic parameters
A total of 48 male Sprague–Dawley rats weighing 200–220 g (Charles
River, L’Arbresle, France) were randomly assigned for 12 weeks to
three different diets. One group was fed a starch-based regular rat
chow, containing 0.64% NaCl (Usine Alimentation Rationnelle,
Villemoisson sur Orge, France) and served as the NSC group. The
two other groups were fed a 60% fructose diet containing either
an amount of sodium similar to the control group and called NSF
(0.64% NaCl) or totally deprived of sodium and named LSF
(o0.01% NaCl).
In the last week of experiment, all rats were housed in individual
cages, and body weight, food, and water consumption, as well as
urinary excretion of water, sodium, and potassium, were measured
daily during 3 days. Urinary excretion of albumin was determined
on 24-h urine collection (AssayMax Rat, Albumin Elisa Kit,
ERA2201-1; Assaypro, Euromedex Souffelweyersheim, France).
Urine concentrations of sodium and potassium were measured by
flame photometry, and plasma concentration of creatinine was
estimated by colorimetric method (Jaffe method). Uric acid was
measured (QuantiChrom Uric Acid Assay Kit; Gentaur, Brussels,
Belgium) using plasma samples from fasting rats at the end of the
studies.
Blood pressure and glucose metabolism
In half of rats, blood pressure was measured at the end of experi-
ments in conscious animals using the tail-cuff method (Narco
Biosystems, Houston, TX). Rats were then fasted (5 h) for the
insulin tolerance test. Glucose was measured in tail blood before and
15, 30, and 75 min after an injection of lispro insulin (0.6 U/kg
intraperitoneal, Humalog; Lilly, Neuilly Sur Seine, France). Because
of its marked fall, blood glucose monitoring was ended at 75 min,
and glucose was given intraperitoneally for rapid recovery.
After 2 days, a catheter was inserted into the left carotid artery
and left under the skin at the dorsum of the neck. After complete
recovery from surgery, rats were fasted overnight, and the catheter
was externalized, thus allowing direct measurements of arterial
pressure and intraperitoneal glucose tolerance test in freely moving
rats. A 40% glucose solution was injected (2 g per kg body weight ,
intraperitoneal) and blood samples were collected before and 15, 30,
60, and 90 min after glucose administration for plasma glucose
and insulin concentration determination (ELISA kit; Millipore,
Billerica, MA).
The homeostasis model assessment of insulin resistance
(HOMA-IR) was calculated using the following equation: HOMA-
IR ¼(fasting blood glucose in mg/dlfasting plasma insulin in
mU/ml)/2.430.53
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C Oudot et al.: Sodium, insulin resistance, and kidney basic research
Renal function and morphology
In half of rats, glomerular filtration rate was estimated by measuring
the rate of clearance of inulin. Rats were anesthetized, and blood was
sampled (200 ml) for measurement of serum concentration of
creatinine. A 15% inulin solution was continuously infused (30 ml/
min) during three 30-min periods of urine collection. Plasma and
urine concentration of inulin was measured using the anthrone–
sulfuric acid technique at 620 nm (Sunrise; Tecan, Lyon, France).
The right kidney was removed, weighed, and prepared for
transmission electron microscopy in the ‘Centre de Ressources en
Imagerie Cellulaire’ (Montpellier, France) as previously described.54
Five fields per glomerulus and two glomeruli per rat were analyzed
for filtration barrier measures. The left kidney was perfusion-fixed
with 10% buffered formalin and blocked in paraffin wax as
previously described.55 Glomerular and tubular size was evaluated
in periodic acid–Schiff sections using the ImageJ software (National
Institutes of Health image, Bethesda, MD). Tubular size, number of
tubular epithelial cells/tubules, and tubular epithelial cell area were
determined as described elsewhere.22 The Bowman’s space was
calculated by subtracting the glomerular area from the glomerular
tuft area. At least 100 glomeruli and 100 tubules were examined for
each rat. Collagen volume fraction was determined by the area
stained with Sirius red (0.1%) in a given field and expressed as a
percentage of the total area within the field. In all, 10 to 15 fields
were analyzed for glomerular, interstitial, and perivascular fibrosis
determination.
Desmin staining was used as a marker of damaged podocytes.56
Paraffin-embedded renal tissue was deparaffinized and blocked for
endogenous peroxidases. Sections were incubated with the primary
anti-desmin antibody (Dako, Trappes, France) diluted 1:300 at
41C overnight. Antibody distribution was visualized by a
streptavidin–biotin complex assay and a DAB substrate kit (Dako).
Sections incubated without primary antibody were used as negative
control. Results were expressed in percentage of glomerular tuft.
Determination of cytokines and NADPH oxidase activity in
the kidney
For measurements of cytokines, a kidney sample was prepared as a
10% homogenate, centrifuged at 9000 gat 4 1C for 30 min, and the
supernatant was harvested. TNF-aand IL-6 were measured using an
ELISA kit (R&D Systems, Minneapolis, MN) in accordance with the
manufacturer’s instructions. The intraassay and interassay variability
of the TNF-aassay was 2.5% and 10.0%, respectively. The intraassay
and interassay variability of the IL-6 assay was 2.9% and 9.2%,
respectively.
NADPH oxidase activity in the kidney homogenates was assessed
with the lucigenin-derived chemiluminescence assay.57 The reaction
was started by the addition of NADPH (0.1 mmol/l) to a suspension
(250 ml) containing the sample (50 ml), lucigenin (5 mmol/l), and
assay phosphate buffer. Luminescence was measured every 30 s for
30 min (luminometer; Tecan). Activity of the enzyme was evaluated
by the slope of O2
1
production, which represents the velocity of
the activity and by the cumulative O2
1
production, expressed as
relative light units per mg tissue.
Adipose tissue
Retroperitoneal and epidydimal adipose tissue was removed,
weighed, and fixed in formalin. Adipocyte size was determined on
sections stained with hematoxylin and eosin. Adipocyte area was
measured in five different sections in each sample.
Macrophage infiltration
ED1 staining was used as a marker of rat macrophages. ED1
immunostaining was performed in renal and adipose tissues as
described above for desmin immunostaining (primary anti-ED1
antibody diluted 1:100; Dako).
Statistical analysis
Results were expressed as means±s.e. and analyzed by one-factor
analysis of variance. Differences between groups were assessed by
Fisher’s protected least significant difference test with a significance
level set for Po0.05. Because of a skewed distribution, albuminuria
was log-transformed before comparison between groups.
DISCLOSURE
All the authors declared no competing interests.
ACKNOWLEDGMENTS
We are grateful to Chantal Cazevieille and Ce
´cile Sanchez for their
technical assistance and interpreting data concerning ultrastructural
evaluation. CO and this research were supported by ‘La Fondation de
Recherche sur l’Hypertension Arte
´rielle.’
REFERENCES
1. Isomaa B, Almgren P, Tuomi T et al. Cardiovascular morbidity and
mortality associated with the metabolic syndrome. Diabetes Care 2001;
24: 683–689.
2. Morris AD, Connel JM. Insulin resistance and essential hypertension:
mechanisms and clinical implications. Am J Med Sci 1994; 307: S47–S52.
3. Reaven GM. Banting lecture 1988. Role of insulin resistance in human
disease. Diabetes 37: 1595–1607.
4. Elliott SS, Keim NL, Stern JS et al. Fructose, weight gain, and the insulin
resistance syndrome. Am J Clin Nutr 1988; 76: 911–922.
5. Delbosc S, Paizanis E, Magous R et al. Involvement of oxidative stress and
NADPH oxidase activation in the development of cardiovascular
complications in a model of insulin resistance, the fructose-fed rat.
Atherosclerosis 2005; 179: 43–49.
6. Nakagawa T, Hu H, Zharikov S et al. A causal role for uric acid in fructose-
induced metabolic syndrome. Am J Physiol 2006; 290: F625–F631.
7. Chen J, Muntner P, Hamm LL et al. The metabolic syndrome and chronic
kidney disease in U.S. adults. Ann Intern Med 2004; 140: 167–174.
8. Sa
´nchez-Lozada LG, Tapia E, Jime
´nez A et al. Fructose-induced metabolic
syndrome is associated with glomerular hypertension and renal
microvascular damage in rats. Am J Physiol Renal Physiol 2007; 292:
F423–F429.
9. Kizhner T, Werman MJ. Long-term fructose intake: biochemical
consequences and altered renal histology in the male rat. Metabolism
2002; 51: 1538–1547.
10. Zaoui P, Rossini E, Pinel N et al. High fructose-fed rats: a model of
glomerulosclerosis involving the renin-angiotensin system and renal
gelatinases. Ann NY Acad Sci 1999; 878: 716–719.
11. Navarro-Gonza
´lez JF, Mora-Ferna
´ndez C. The role of inflammatory
cytokines in diabetic nephropathy. J Am Soc Nephrol 2008; 19: 433–442.
12. Lazar MA. The humoral side of insulin resistance. Nat Med 2006; 12: 43–44.
13. Knight SF, Yuan J, Roy S et al. Simvastatin and tempol protect against
endothelial dysfunction and renal injury in a model of obesity and
hypertension. Am J Physiol Renal Physiol 2010; 298: F86–F94.
14. Busserolles J, Gueux E, Rock E et al. Substituting honey for refined
carbohydrates protects rats from hypertriglyceridemic and prooxidative
effects of fructose. J Nutr 2002; 132: 3379–3382.
15. Brown IJ, Stamler J, Van Horn L et al. International Study of Macro/
Micronutrients and Blood Pressure Research Group. Sugar-sweetened
beverage, sugar intake of individuals, and their blood pressure:
international study of macro/micronutrients and blood pressure.
Hypertension 2011; 57: 695–701.
16. Sechi LA. Mechanisms of insulin resistance in rat models of hypertension
and their relationships with salt sensitivity. J Hypertens 1999; 17:
1229–1237.
17. Johnson MD, Zhang HY, Kotchen TA. Sucrose does not raise blood
pressure in rats maintained on a low salt intake. Hypertension 1993; 6:
779–785.
682 Kidney International (2013) 83,674–683
basic research C Oudot et al.: Sodium, insulin resistance, and kidney
18. Rugale C, Delbosc S, Cristol JP et al. Sodium restriction prevents cardiac
hypertrophy and oxidative stress in angiotensin II hypertension. Am J
Physiol Heart Circ Physiol 2003; 284: H1744–H1750.
19. Sharma K, Ramachandrarao S, Qiu G et al. Adiponectin regulates
albuminuria and podocyte function in mice. J Clin Invest 2008; 118:
1645–1656.
20. Tesauro M, Canale MP, Rodia G et al. Metabolic syndrome, chronic kidney,
and cardiovascular diseases: role of adipokines. Cardiol Res Pract 2011;
2011; 1–11.
21. Mimran A, du Cailar G. Dietary sodium: the dark horse amongst
cardiovascular and renal risk factors. Nephrol Dial Transplant 2008; 23:
2138–2141.
22. Nakayama T, Kosugi T, Gersch M et al. Dietary fructose causes
tubulointerstitial injury in the normal rat kidney. Am J Physiol Renal
Physiol 2010; 298: F712–F720.
23. Cosenzi A, Bernobich E, Plazzotta N et al. Lacidipine reduces high blood
pressure and the target organ damage induced by high fructose diet in
rats. J Hypertens 1999; 17: 965–971.
24. Sa
´nchez-Lozada LG, Tapia E, Bautista-Garcı
´aPet al. Effects of febuxostat
on metabolic and renal alterations in rats with fructose-induced
metabolic syndrome. Am J Physiol Renal Physiol 2008; 294: F710–F718.
25. Chang JW, Kim CS, Kim SB et al. Proinflammatory cytokine-induced NF-
kappaB activation in human mesangial cells is mediated through
intracellular calcium but not ROS: effects of silymarin. Nephron Exp
Nephrol 2006; 103: e156–e165.
26. Pirotzky E, Delattre RM, Hellegouarch A et al. Interleukin-6 production by
tumor necrosis factor and lipopolysaccharide-stimulated rat renal cells.
Clin Immunol Immunopathol 1990; 56: 271–279.
27. Coleman DL, Ruef C. Interleukin-6: an autocrine regulator of mesangial
cell growth. Kidney Int 1992; 41: 604–606.
28. DiPetrillo K, Gesek FA. Pentoxifylline ameliorates renal tumor necrosis
factor expression, sodium retention, and renal hypertrophy in diabetic
rats. Am J Nephrol 2004; 24: 352–359.
29. McCarthy E, Sharma R, Sharma M et al. TNF-alpha increases albumin
permeability of isolated rat glomeruli through the generation of
superoxide. J Am Soc Nephrol 1998; 9: 433–438.
30. Catena C, Cavarape A, Novello M et al. Insulin receptors and renal sodium
handling in hypertensive fructose-fed rats. Kidney Int 2003; 64:
2163–2171.
31. Henriksen EJ. Improvement of insulin sensitivity by antagonism of the
renin-angiotensin system. Am J Physiol Regul Integr Comp Physiol 2007;
293: R974–R980.
32. Perkins JM, Davis SN. The renin-angiotensin-aldosterone system: a pivotal
role in insulin sensitivity and glycemic control. Curr Opin Endocrinol
Diabetes Obes 2008; 15: 147–152.
33. Wei Y, Sowers JR, Clark SE et al. Angiotensin II-induced skeletal muscle
insulin resistance mediated by NF-kappaB activation via NADPH oxidase.
Am J Physiol Endocrinol Metab 2008; 294: E345–E351.
34. Chamarthi B, Williams GH, Ricchiuti V et al. Inflammation and
hypertension: the interplay of interleukin-6, dietary sodium, and the
renin-angiotensin system in humans. Am J Hypertens 2011; 24:
1143–1148.
35. Hayden MR, Whaley-Connell A, Sowers JR. Renal redox stress and
remodeling in metabolic syndrome, type 2 diabetes mellitus, and diabetic
nephropathy: paying homage to the podocyte. Am J Nephrol 2005; 25:
553–569.
36. Whaley-Connell A, DeMarco VG, Lastra G et al. Insulin resistance, oxidative
stress, and podocyte injury: role of rosuvastatin modulation of filtration
barrier injury. Am J Nephrol 2008; 28: 67–75.
37. Bonnet F, Cooper ME, Kawachi H et al. Irbesartan normalises the
deficiency in glomerular nephrin expression in a model of diabetes and
hypertension. Diabetologia 2001; 44: 874–877.
38. Hajer GR, van Haeften TW, Visseren FL. Adipose tissue dysfunction in
obesity, diabetes, and vascular diseases. Eur Heart J 2008; 29: 2959–2971.
39. Blu
¨her M. Adipose tissue dysfunction in obesity. Exp Clin Endocrinol
Diabetes 2009; 117: 241–250.
40. Greenberg AS, Obin MS. Obesity and the role of adipose tissue in
inflammation and metabolism. Am J Clin Nutr 2006; 83: 461S–465S.
41. Shapiro A, Mu W, Roncal C et al. Fructose-induced leptin resistance
exacerbates weight gain in response to subsequent high-fat feeding. Am
J Physiol Regul Integr Comp Physiol 2008; 295: R1370–R1375.
42. Weisberg SP, McCann D, Desai M et al. Obesity is associated with
macrophage accumulation in adipose tissue. J Clin Invest 2003; 112:
1796–1808.
43. Xu H, Barnes GT, Yang Q et al. Chronic inflammation in fat plays a crucial
role in the development of obesity-related insulin resistance. J Clin Invest
2003; 112: 1821–1830.
44. Suganami T, Nishida J, Ogawa Y. A paracrine loop between adipocytes
and macrophages aggravates inflammatory changes: role of free fatty
acids and tumor necrosis factor alpha. Arterioscler Thromb Vasc Biol 2005;
25: 2062–2068.
45. Whaley-Connell AT, Chowdhury NA, Hayden MR et al. Oxidative stress
and glomerular filtration barrier injury: role of the renin-angiotensin
system in the Ren2 transgenic rat. Am J Physiol Renal Physiol 2006; 291:
F1308–F1314.
46. Johnson RJ, Sanchez-Lozada LG, Nakagawa T. The effect of fructose on
renal biology and disease. J Am Soc Nephrol 2010; 21: 2036–2039.
47. Singh AK, Amlal H, Haas PJ et al. Fructose-induced hypertension: essential
role of chloride and fructose absorbing transporters PAT1 and GLUT5.
Kidney Int 2008; 74: 438–447.
48. Egan BM, Weder AB, Petrin J et al. Neurohumoral and metabolic effects of
short-term dietary NaCl restriction in men. Relationship to salt-sensitivity
status. Am J Hypertens 1991; 4: 416–421.
49. Fliser D, Fode P, Arnold U et al. The effect of dietary salt on insulin
sensitivity. Eur J Clin Invest 1995; 25: 39–43.
50. Prada P, Okamoto MM, Furukawa LN et al. High- or low-salt diet from
weaning to adulthood: effect on insulin sensitivity in Wistar rats.
Hypertension 2000; 35: 424–429.
51. Donovan DS, Solomon CG, Seely EW et al. Effect of sodium intake on
insulin sensitivity. Am J Physiol 1993; 264: E730–E734.
52. Hayden MR, Sowers JR. Childhood-adolescent obesity in the cardiorenal
syndrome: lessons from animal models. Cardiorenal Med 2011; 1: 75–86.
53. Cacho J, Sevillano J, de Castro J et al. Validation of simple indexes to
assess insulin sensitivity during pregnancy in Wistar and Sprague-Dawley
rats. Am J Physiol Endocrinol Metab 2008; 295: 1269–1276.
54. Bouttier M, Saumet A, Peter M et al. Retroviral GAG proteins recruit AGO2
on viral RNAs without affecting RNA accumulation and translation.
Nucleic Acids Res 2012; 40: 775–786.
55. Casellas D, Herizi A, Artuso A et al. Candesartan prevents L-NAME-
induced cardiorenal injury in spontaneously hypertensive rats beyond
hypotensive effects. J Renin Angiotensin Aldosterone Syst 2001; 2: S84–S90.
56. Toyonaga J, Tsuruya K, Ikeda H et al. Spironolactone inhibits
hyperglycemia-induced podocyte injury by attenuating ROS production.
Nephrol Dial Transplant 2011; 26: 2475–2484.
57. Sedeek M, Callera G, Montezano A et al. Critical role of Nox4-based
NADPH oxidase in glucose-induced oxidative stress in the kidney:
implications in type 2 diabetic nephropathy. Am J Physiol Renal Physiol
2010; 299: F1348–F1358.
Kidney International (2013) 83, 674–683 683
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