Simvastatin and tempol protect against endothelial dysfunction and renal
injury in a model of obesity and hypertension
Sarah F. Knight,1Jianghe Yuan,1Siddhartha Roy,1and John D. Imig2
1Vascular Biology Center, Medical College of Georgia, Augusta, Georgia; and2Department of Pharmacology, Medical
College of Wisconsin, Milwaukee, Wisconsin
Submitted 21 June 2009; accepted in final form 4 November 2009
Knight SF, Yuan J, Roy S, Imig JD. Simvastatin and tempol protect
against endothelial dysfunction and renal injury in a model of obesity and
hypertension. Am J Physiol Renal Physiol 298: F86–F94, 2010. First
published November 11, 2009; doi:10.1152/ajprenal.00351.2009.—
Obesity and hypertension are risk factors for the development of
chronic kidney disease. The mechanisms by which elevated blood
pressure and fatty acids lead to the development of renal injury are
incompletely understood. Here, we investigated the contributions of
cholesterol and oxidative stress to renal endothelial dysfunction and
glomerular injury in a model of obesity and hypertension. Male
Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR)
were fed a normal diet, a high-fat diet, a high-fat diet with tempol, or
a high-fat diet with simvastatin for up to 10 wk. Blood pressure was
not altered by a high-fat diet or treatments. After 3 wk, renal afferent
dilatory responses to acetylcholine were impaired in WKY rats and
SHR fed a high-fat diet. Tempol treatment prevented this vascular
dysfunction in both strains; however, simvastatin treatment demon-
strated greater beneficial effects in the SHR. Albuminuria was observed in
the SHR and was exacerbated by a high-fat diet. Tempol and simvastatin
treatment significantly ameliorated albuminuria in the SHR fed a high-fat
diet. Ten weeks on a high-fat resulted in an increase in urinary
8-isoprostane in WKY rats and SHR, and tempol and simvastatin
treatment prevented this increase, indicating a reduction in renal
oxidative stress. Monocyte chemoattractant protein-1 (MCP-1) excre-
tion was significantly elevated by a high-fat diet in both strains, and
tempol prevented this increase. Interestingly, simvastatin treatment
had no effect on MCP-1 levels. These data indicate that tempol and
simvastatin treatment via a reduction in oxidative stress improve renal
endothelial function and decrease glomerular injury in a model of
obesity and hypertension.
kidney; oxidative stress; metabolic syndrome; inflammation
OBESITY HAS REACHED EPIDEMIC proportions in the developed
world, with 50% of the US population now classified as
overweight or obese, and the numbers are rising (National
Health and Nutrition Examination Survey, available at http://
www.cdc.gov/nchs/nhanes.htm). Obesity is associated with in-
creased risk of developing diabetes, hypercholesterolemia, and
hypertension, which together are described as the metabolic
syndrome (18, 29). Independently, obesity and hypertension
increase the risk of renal dysfunction; obese patients are four
times more likely to develop renal disease than nonobese
patients, and hypertension accounts for 25% of renal dysfunc-
tion cases (27). Obese patients are at a higher risk of develop-
ing hypertension, therefore combining risk factors and com-
pounding the negative effect on cardiovascular health. In
particular, renal injury associated with obesity and hyperten-
sion has been shown to be more severe than renal disease
observed as a result of each risk factor alone. This additive
effect has also been observed in animal models of obesity with
hypertension such as the SHR/NDmcr-cp, a rat model of
metabolic syndrome and the spontaneously hypertensive rat
(SHR) fed a high-fat diet (26, 32).
Obesity and hypertension have both been associated with
elevated levels of circulating oxidative metabolites, such as
superoxide. Increased levels of oxidative stress in both obese
and hypertensive patients are thought to contribute to chronic
kidney disease; however, the extent to which blood pressure
and oxidative stress contribute to the progression of renal
vascular dysfunction, inflammation, and injury remains unclear
(12, 34). In hypertensive animal models such as the SHR and
angiotensin hypertension, elevated levels of superoxide have
been detected (24). These elevated oxidative molecules may
contribute to some of the characteristic pathological changes
that occur in the SHR, such as renal morphological alterations
as hypertension develops (1, 28). Because hypertension is
associated with increased superoxide production, we were
interested in whether hypertension combined with a high-fat
diet increases the susceptibility or severity of renal injury and
the contribution of superoxide and cholesterol to this injury.
This is the first time that the effect of tempol and simvastatin
on endothelial function and kidney injury has been studied in
an animal model of essential hypertension with obesity. In-
creased production of superoxide can overpower endogenous
scavenging molecules such as superoxide dismutase and in-
duce renal dysfunction by way of a number of mechanisms, for
example, reducing the bioavailability of vasodilators such as
nitric oxide (35, 36). We have previously observed no increase
in blood pressure in WKY rats as a result of 10-wk high-fat
feeding; however, in a clinical setting hypercholesterolemia is
often present in combination with hypertension (26). There-
fore, in this study we use the rat model of essential hyperten-
sion, the SHR fed a-high fat diet, to explore the contributions
of obesity and hypertension to renal injury. High levels of renal
vascular oxidative stress have been identified in animal models
of obesity such as the leptin-deficient mouse, the db/db and the
leptin receptor-deficient rat model, the obese Zucker rat, as
well as in obese humans (2, 4, 16). One reason for the
increased production of superoxide in obesity could be as a
result of elevated levels of cholesterol, which has also been
associated with obesity and increased risk of developing car-
diovascular disease (20) (http://www.americanheart.org/
presenter.jhtml?identifier?4639). High cholesterol levels have
been shown to contribute to elevated superoxide generation;
therefore, high cholesterol levels could contribute to the renal
dysfunction frequently observed in obesity (10).
Address for reprint requests and other correspondence: J. D. Imig, Dept. of
Pharmacology, Medical College of Wisconsin, Madison, WI (e-mail: jdimig
Am J Physiol Renal Physiol 298: F86–F94, 2010.
First published November 11, 2009; doi:10.1152/ajprenal.00351.2009.
0363-6127/10 $8.00 Copyright © 2010 the American Physiological Society http://www.ajprenal.orgF86
We hypothesized that in an animal model of obesity and
hypertension, the increased severity of renal injury observed in
SHR as a result of a high-fat diet is a result of elevated
cholesterol levels combined with preexisting oxidative stress as
a result of the hypertension. Elevated levels of circulating
cholesterol give rise to increased production of superoxide,
which could contribute to endothelial dysfunction and renal
injury that are characteristic of this model of obesity. 3-Hy-
droxy-3-methylglutaryl-coenzyme A (HMG -CoA) reductase
inhibitors or statins are hypolipidemic drugs used for the
treatment of high cholesterol in those at risk for the develop-
ment of cardiovascular disease. HMG -CoA reductase is the
rate-limiting step in the production of cholesterol, and statins
have been shown to reduce “bad” LDL cholesterol levels from
up to 30–50% (3). In addition to the cholesterol-lowering
effects of statins, other non-lipid-mediated beneficial pleiotro-
pic effects have been observed, such as improved endothelial
function and antioxidative effects (5, 13, 23). Therefore, in this
study we used simvastatin, an HMG-CoA reductase inhibitor,
and 4-hydroxy-tempol (tempol), a superoxide dismutase mi-
metic, to explore the contribution of cholesterol and oxidative
stress to afferent arteriole endothelial dysfunction and renal
injury, in a model of obesity and hypertension.
MATERIALS AND METHODS
All animal studies were approved by the Medical College of
Georgia institutional review committee according to the National
Institutes of Health guidelines for the care and use of laboratory
animals. Eight-week-old male Wistar-Kyoto (WKY) rats and SHR
were purchased from Charles River Laboratories (Wilmington, MA)
and were divided into four groups: A–D (n ? 5–6). Group A were fed
normal rat chow containing 7% fat (Teklad), and the remaining
groups, groups B–D, received a high-fat diet containing 36% fat (no.
F2685, BioServ, Frenchtown, NJ) ad libitum for 10 wk. Group C
received 1 mmol tempol in their drinking water, and group D received
simvastatin (10 mg?kg?1?day?1) in their drinking water throughout
the 10-wk study period. After an initial training period, rats were
weighed and systolic blood pressure was measured by tail-cuff pleth-
ysmography every 7 days, as previously demonstrated (26).
In Vitro Perfused Juxtamedullary Nephron Experiments
Experiments were conducted in vitro using the perfused jux-
tamedullary nephron technique, as previously described (26). Male
WKY rats and SHR (n ? 4) were anesthetized with pentobarbital
sodium (40 mg/kg body wt ip). The right renal artery was cannu-
lated and perfused with a Tyrode buffer solution containing 5.2%
BSA and a complement of L-amino acids. The kidney was removed
and sectioned along the longitudinal axis, with care taken to leave
the papilla intact on the dorsal two-thirds of the kidney. The
vasculature was isolated as previously described (26). The perfus-
ate was consistently perfused with 95% O2-5% CO2. Perfusion
pressure was set at 110 mmHg and monitored continuously. The
inner cortical surface of the kidney was superfused with warmed
(37°C) Tyrode buffer containing 1% BSA. Vessel inner diameters
were viewed by video microscopy and measured using an image-
shearing monitor. An afferent arteriole was selected, and after a
20-min equilibration the vessel was constricted using 1 mmol/l
phenylephrine added to the superfusate. The vessel diameter was
measured and recorded as the baseline. Acetylcholine was added to
the perfusate to make a final concentration of 1 ? 10?8mmol/l;
then, the concentration of acetylcholine was increased to 1 ? 10?7
mmol, 1 ? 10?6, and finally 1 ? 10?5mmol/l. Mean vessel
diameter was recorded for 15 min at each concentration of acetyl-
choline. The vessel was perfused with 1% BSA for 15 min in the
absence of acetylcholine, followed by a 15-min incubation with
sodium nitroprusside to exclude the contribution of the smooth
muscle to any differences in dilatory response to acetylcholine.
In Vitro Assays and Enzyme-Linked Immunoassays
Rats were housed in metabolic cages for 24 h at the end of the
10-wk experiment to collect urine for analysis. Plasma-free choles-
terol levels were measured using a commercially available kit from
Wako Diagnostics (Richmond VA). Urinary microalbumin levels
were measured by enzyme-linked immunoassay (ELISA) using a
commercial kit from SPI-bio (Paris, France). Urinary monocyte che-
moattractant protein-1 (MCP-1) and 8-isoprostane levels were also
measured by commercial ELISA (BD Biosciences, San Jose, CA and
Cayman, Ann Arbor, MI).
Five-micrometer frozen kidney sections were cut and incubated
overnight at room temperature with mouse anti-rat CD68 primary
antibody (1:100, Serotec, Raleigh, NC) followed by the secondary
antibody goat anti-mouse IgG HRP (1:50, Serotec) for 1 h at room
temperature. Slides were incubated with AEC substrate chromogen
(DAKO, Carpinteria, CA) for 20 min, rinsed, and counterstained with
Mayers hematoxylin for 30 s. Photographs were taken at ?400
magnification. Eight randomly selected glomeruli were photographed
from each of 6 kidneys per treatment group, totaling 48 images. A
blinded reviewer counted the number of CD68-positive cells. Group
means and SEs of positively stained cells were calculated per square
Five-micrometer frozen kidney sections were incubated over-
night at room temperature with goat anti-human nephrin primary
antibody (1:50, sc-19000, Santa Cruz Biotechnology, Santa Cruz,
CA) followed by rabbit anti-goat Cy-3 fluorescent-tagged second-
ary antibody (1:400, for 1 h, Zymed). Slides were mounted using
Prolong Gold anti-fade (Invitrogen) and glass coverslips. Photo-
graphs were taken at ?400 magnification. Ten randomly selected
glomeruli were photographed from each of 6 kidneys per treatment
group, totaling 60 images/group. Fluorescent intensity of glomeruli
was measured using Metamorph software (Molecular Devices),
correcting for background fluorescence, and means and SEs were
All statistical analysis of data was performed using 2-way ANOVA
and the Bonferroni posttest. Differences are considered statistically
significant if P values are 0.05 or below.
Body Weight and Blood Pressure
We measured mean body weight in all groups at baseline
and throughout the study. Figure 1, A and B, displays the mean
body weight of the WKY and SHR fed a normal diet, a high-fat
diet, a high-fat diet with tempol in their drinking water, or a
high-fat diet with simvastatin in their drinking water. We
observed that after 10 wk of high-fat feeding, the WKY rats
had a slightly higher body weight than the SHR; however, this
did not reach significance. Interestingly, the WKY rats receiv-
ing a high-fat diet with tempol or simvastatin treatment dis-
played a significant increase in body weight compared with
those fed a normal diet (P ? 0.05). Figure 1B shows data for
the SHR where 10 wk on a high-fat diet induced a significant
SIMVASTATIN AND TEMPOL IN RENAL INJURY ASSOCIATED WITH OBESITY AND HYPERTENSION
AJP-Renal Physiol • VOL 298 • JANUARY 2010 • www.ajprenal.org
inflammation and fibrosis in stroke-prone rats. Am J Pathol 170: 1165–
20. Huang J, Parish R, Mansi I, Yu H, Kennen EM, Davis T, Carden D.
Non-high-density lipoprotein cholesterol in patients with metabolic syn-
drome. J Investig Med 56: 931–936, 2008.
21. Ichinose K, Maeshima Y, Yamamoto Y, Kinomura M, Hirokoshi K,
Kitayama H, Takazawa Y, Sugiyama H, Yamasaki Y, Agata N,
Makino H. 2-(8-Hydroxy-6-methoxy-1-oxo-1h-2-benzopyran-3-yl) pro-
pionic acid, an inhibitor of angiogenesis, ameliorates renal alterations in
obese type 2 diabetic mice. Diabetes 55: 1232–1242, 2006.
22. Ivanovski O, Szumilak D, Nguyen-Khoa T, Nikolov IG, Joki N, Mothu
N, Maizel J, Westenfeld R, Ketteler M, Lacour B, Drueke TB, Massy
ZA. Effect of simvastatin in apolipoprotein E deficient mice with surgi-
cally induced chronic renal failure. J Urol 179: 1631–1636, 2008.
23. Jasinska M, Owczarek J, Orszulak-Michalak D. Statins: a new insight
into their mechanisms of action and consequent pleiotropic effects. Phar-
macol Rep 59: 483–499, 2007.
24. Just A, Olson AJ, Whitten CL, Arendshorst WJ. Superoxide mediates
acute renal vasoconstriction produced by angiotensin II and cat-
echolamines by a mechanism independent of nitric oxide. Am J Physiol
Heart Circ Physiol 292: H83–H92, 2007.
25. Kagiyama S, Tsuchihashi T, Abe I, Matsumura K, Fujishima M.
Central infusion of L-arginine or superoxide dismutase does not alter
arterial pressure in SHR. Hypertens Res 23: 339–343, 2000.
26. Knight SF, Quigley JE, Yuan J, Roy SS, Elmarakby A, Imig JD.
Endothelial dysfunction and the development of renal injury in spontane-
ously hypertensive rats fed a high-fat diet. Hypertension 51: 352–359,
27. Kramer H, Luke A, Bidani A, Cao G, Cooper R, McGee D. Obesity
and prevalent and incident CKD: the Hypertension Detection and Fol-
low-Up Program. Am J Kidney Dis 46: 587–594, 2005.
28. Lazaro A, Gallego-Delgado J, Justo P, Esteban V, Osende J, Mezzano
S, Ortiz A, Egido J. Long-term blood pressure control prevents oxidative
renal injury. Antioxid Redox Signal 7: 1285–1293, 2005.
29. Lind L, Berne C, Lithell H. Prevalence of insulin resistance in essential
hypertension. J Hypertens 13: 1457–1462, 1995.
30. Macconi D, Bonomelli M, Benigni A, Plati T, Sangalli F, Longaretti L,
Conti S, Kawachi H, Hill P, Remuzzi G, Remuzzi A. Pathophysiologic
implications of reduced podocyte number in a rat model of progressive
glomerular injury. Am J Pathol 168: 42–54, 2006.
31. Miyata K, Ohashi N, Suzaki Y, Katsurada A, Kobori H. Sequential
activation of the reactive oxygen species/angiotensinogen/renin-angioten-
sin system axis in renal injury of type 2 diabetic rats. Clin Exp Pharmacol
Physiol 35: 922–927, 2008.
32. Nagase M, Yoshida S, Shibata S, Nagase T, Gotoda T, Ando K, Fujita
T. Enhanced aldosterone signaling in the early nephropathy of rats with
metabolic syndrome: possible contribution of fat-derived factors. J Am Soc
Nephrol 17: 3438–3446, 2006.
33. Naito M, Shenoy A, Aoyama I, Koopmeiners JS, Komers R, Schnaper
HW, Bomsztyk K. High ambient glucose augments angiotensin ii-in-
duced proinflammatory gene mRNA expression in human mesangial cells:
effects of valsartan and simvastatin. Am J Nephrol 30: 99–111, 2009.
34. Nangaku M, Izuhara Y, Usuda N, Inagi R, Shibata T, Sugiyama S,
Kurokawa K, van Ypersele de Strihou C, Miyata T. In a type 2 diabetic
nephropathy rat model, the improvement of obesity by a low calorie diet
reduces oxidative/carbonyl stress and prevents diabetic nephropathy.
Nephrol Dial Transplant 20: 2661–2669, 2005.
35. Peixoto EB, Pessoa BS, Biswas SK, Lopes de Faria JB. Antioxidant
SOD mimetic prevents NADPH oxidase-induced oxidative stress and
renal damage in the early stage of experimental diabetes and hypertension.
Am J Nephrol 29: 309–318, 2008.
36. Rafikova O, Salah EM, Tofovic SP. Renal and metabolic effects of
tempol in obese ZSF1 rats—distinct role for superoxide and hydrogen
peroxide in diabetic renal injury. Metabolism 57: 1434–1444, 2008.
37. Rodriguez-Iturbe B, Vaziri ND, Herrera-Acosta J, Johnson RJ. Oxi-
dative stress, renal infiltration of immune cells, and salt-sensitive hyper-
tension: all for one and one for all. Am J Physiol Renal Physiol 286:
38. Sarafidis PA. Obesity, insulin resistance and kidney disease risk: insights
into the relationship. Curr Opin Nephrol Hypertens 17: 450–456, 2008.
39. Schnackenberg CG, Welch WJ, Wilcox CS. Normalization of blood
pressure and renal vascular resistance in SHR with a membrane-permeable
superoxide dismutase mimetic: role of nitric oxide. Hypertension 32:
40. Stewart T, Jung FF, Manning J, Vehaskari VM. Kidney immune cell
infiltration and oxidative stress contribute to prenatally programmed hy-
pertension. Kidney Int 68: 2180–2188, 2005.
41. Wang D, Chen Y, Chabrashvili T, Aslam S, Borrego Conde LJ,
Umans JG, Wilcox CS. Role of oxidative stress in endothelial dysfunc-
tion and enhanced responses to angiotensin II of afferent arterioles from
rabbits infused with angiotensin II. J Am Soc Nephrol 14: 2783–2789,
42. Wang J, Zou H, Li Q, Wang Y, Xu Q. The expression of monocyte
chemoattractant protein-1 and C-C chemokine receptor 2 in post-kidney
transplant patients and the influence of simvastatin treatment. Clin Chim
Acta 373: 44–48, 2006.
43. Weisberg SP, McCann D, Desai M, Rosenbaum M, Leibel RL, Fer-
rante AW Jr. Obesity is associated with macrophage accumulation in
adipose tissue. J Clin Invest 112: 1796–1808, 2003.
44. Whaley-Connell A, Habibi J, Nistala R, Cooper SA, Karuparthi PR,
Hayden MR, Rehmer N, DeMarco VG, Andresen BT, Wei Y, Ferrario
C, Sowers JR. Attenuation of NADPH oxidase activation and glomerular
filtration barrier remodeling with statin treatment. Hypertension 51: 474–
45. Wilcox CS, Pearlman A. Chemistry and antihypertensive effects of
tempol and other nitroxides. Pharmacol Rev 60: 418–469, 2008.
46. Wu Y, Dong J, Yuan L, Liang C, Ren K, Zhang W, Fang F, Shen J.
Nephrin and podocin loss is prevented by mycophenolate mofetil in early
experimental diabetic nephropathy. Cytokine 44: 85–91, 2008.
47. Yanes L, Romero D, Iliescu R, Cucchiarelli VE, Fortepiani LA,
Santacruz F, Bell W, Zhang H, Reckelhoff JF. Systemic arterial
pressure response to two weeks of Tempol therapy in SHR: involvement
of NO, the RAS, and oxidative stress. Am J Physiol Regul Integr Comp
Physiol 288: R903–R908, 2005.
48. Zhang H, Saha J, Byun J, Schin M, Lorenz M, Kennedy RT, Kretzler
M, Feldman EL, Pennathur S, Brosius FC III. Rosiglitazone reduces
renal and plasma markers of oxidative injury and reverses urinary metabo-
lite abnormalities in the amelioration of diabetic nephropathy. Am J
Physiol Renal Physiol 295: F1071–F1081, 2008.
SIMVASTATIN AND TEMPOL IN RENAL INJURY ASSOCIATED WITH OBESITY AND HYPERTENSION
AJP-Renal Physiol • VOL 298 • JANUARY 2010 • www.ajprenal.org