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Kidney International, Vol. 67 (2005), pp.

237247
Mild hyperuricemia induces vasoconstriction and maintains
glomerular hypertension in normal and remnant kidney rats
LAURA G. S ANCHEZ-LOZADA, EDILIA TAPIA, JOS E SANTAMARA, CARMEN AVILA-CASADO,
VIRGILIA SOTO, TOM AS NEPOMUCENO, BERNARDO RODRGUEZ-ITURBE, RICHARD J. JOHNSON,
and JAIME HERRERA-ACOSTA
Departments of Nephrology and Pathology, Instituto Nacional de Cardiologa Ignacio Chavez, Mexico City, Mexico; Renal Service
and Laboratory, Hospital Universitario and Instituto de Investigaciones Biom edicas, Maracaibo, Venezuela; and Department of
Medicine/Nephrology, University of Florida, Gainesville, Florida
Mild hyperuricemia induces vasoconstriction and maintains
glomerular hypertension in normal and remnant kidney rats.
Background. Hyperuricemia has been associated with renal
disease. Because glomerular hemodynamic alterations critically
contribute to initiation and progression of renal disease, we
evaluated the effect of mild hyperuricemia in glomerular mi-
crocirculatory changes in rats under normal conditions and with
renal injury induced by subtotal renal ablation (RK).
Methods. Hyperuricemia was induced in normal and
remnant kidney (RK) rats on a normal sodium diet by ad-
ministration of oxonic acid (OA). To prevent hyperuricemia,
allopurinol (AP) was administered concomitantly. Glomerular
hemodynamics were evaluated by micropuncture techniques.
Systolic blood pressure (SBP), proteinuria, arterial morphol-
ogy, and serum uric acid were measured. In RK rats, glomeru-
losclerosis, brosis, and inammatory cell inltration (CD5+)
were also assessed.
Results. In normal rats, hyperuricemia resulted in afferent
arteriole thickening associated with renal cortical vasoconstric-
tion[single nephronglomerular ltrationrate (SNGFR) 35%,
P < 0.05) and glomerular hypertension (P < 0.05). Allopuri-
nol treatment prevented structural and functional alterations.
In RK rats, hyperuricemia produced more renal vascular dam-
age than control animals coupled with severe cortical vasocon-
striction (SNGFR 40%, P < 0.05) and persistent glomerular
hypertension. Allopurinol partially prevented cortical vasocon-
striction, and fully prevented arteriolopathy and glomerular hy-
pertensionassociatedwithsignicantlyless inltrationof CD5+
cells.
Conclusion. Hyperuricemia induces arteriolopathy of pre-
glomerular vessels, which impairs the autoregulatory response
of afferent arterioles, resulting in glomerular hypertension. Lu-
men obliteration induced by vascular wall thickening produces
severe renal hypoperfusion. The resulting ischemia is a potent
stimulus that induces tubulointerstitial inammation and bro-
sis, as well as arterial hypertension. These studies provide a
Key words: hyperuricemia, arteriolopathy, cortical vasoconstriction,
glomerular hypertension.
Received for publication May 25, 2004
and in revised form July 2, 2004
Accepted for publication July 27, 2004
C
2005 by the International Society of Nephrology
potential mechanism by which hyperuricemia can mediate hy-
pertension and renal disease.
Hyperuricemia has long been associated with renal dis-
ease. The histologic lesion termed gouty nephropathy
(glomerulosclerosis, interstitial brosis, and renal arte-
riolosclerosis, often with focal interstitial urate crystal
deposition) has been observed in autopsies of 79% to
99% of patients with gout [1]. A rare condition termed
familial juvenile hyperuricemic nephropathy (FJHN) is
characterized by reduced fractional excretion of urate,
hyperuricemia, and renal interstitial lesion often without
deposits of uratecrystals; contrarytogout, this diseasede-
velops in young men, women, and children [2]. Studies in
childrenof these families showedthat hyperuricemia plus
reduced FEurate is found in 50% of apparently healthy
siblings still with normal renal function, which suggests
that uric acid is the causative agent in this nephropathy
[3]. Moreover, in recipients of liver transplant, renal im-
pairment evaluated as serum creatinine was higher in hy-
peruricemic than in nonhyperuricemic patients, and the
peak uric acid correlated with corresponding serumcrea-
tinine (r = 0.7). Treatment with allopurinol for 3 months
reduced serum creatinine (P = 0.01) without changing
the type or dose of immunosuppression [4]. In addition,
clinical studies demonstrated that hyperuricemia is a risk
factor for progression in IgA nephropathy [5, 6] and con-
fers a greater risk than proteinuria for developing chronic
renal disease in the general population [7]. However, the
pathogenic mechanism by which the increment of serum
uric acid may cause renal injury has remained elusive.
Several experimental and clinical studies explored the
possibility that hyperuricemia may induce renal vas-
cular damage. Messerli et al evaluated the effect of
hyperuricemia on renal hemodynamics in normoten-
sive subjects and patients with uncomplicated essen-
tial hypertension. They found that mild asymptomatic
237
238 S anchez-Lozada et al: Mild hyperuricemia, cortical vasoconstriction, and glomerular hypertension
hyperuricemia was associated with diminished renal
blood ow and increased renal vascular resistance re-
gardless of the level of arterial pressure and glomeru-
lar ltration rate. They suggested that the high frequency
of hyperuricemia in the essential hypertensive popula-
tionmost likely reects hypertensive vascular disease and
early nephrosclerosis [8]. In spontaneously hypertensive
rats (SHR) treated with DOCA-salt, Sesoko et al demon-
strated major differences in renal dynamics as compared
with SHR, DOCA-salt, or Wistar-Kyoto (WKY) alone
in association with higher serum uric acid levels. Renal
bloodowwas markedlyreduced, andvascular resistance
index was approximately 467% greater in SHR DOCA-
salt than control SHR. The increase in renal vascular
resistance in these rats correlated with the serum uric
acid, level whereas renal blood ow correlated inversely.
These correlations were not found in the other 3 groups
with normal renal blood ows. Histologic examination
of the kidneys of DOCA-salt SHR revealed remarkable
wall thickening and obstruction of small arteries with b-
rinoid necrosis in the vascular walls. These authors sug-
gested that reduced renal blood ow and hyperuricemia
may share a common mechanism [9].
While the above studies suggest an association of low
renal blood ow and high renal vascular resistance with
hyperuricemia, they do not directly test whether hype-
ruricemia per se can induce these hemodynamic alter-
ations. To do this, one would likely study primary models
of hyperuricemia, but unfortunately, most previous mod-
els of hyperuricemia induced an intense increment of
serum uric acid, with obstruction of renal tubules with
urate crystals resulting in acute urate nephropathy [10,
11]; thus, these models were not appropriate to study the
direct effect of mild hyperuricemia as is seen in gout,
FJHN, and hypertension. In this regard, Mazzali et al re-
cently developed an experimental model of mild hyper-
uricemia in rats by inhibiting uricase with oxonic acid on
a low salt diet. This maneuver produced a 2-fold incre-
ment of serum uric acid, hypertension, tubulointerstitial
injury, and arteriolopathy in normal animals, as well as
exacerbated cyclosporine nephrotoxicity [1214]. These
changes were prevented by allopurinol or the uricosuric
agent benziodarone [1214].
Because glomerular hemodynamic alterations criti-
cally contribute to the initiation and progression of renal
disease, we performed micropuncture studies to evaluate
the effect of mild hyperuricemia on glomerular micro-
circulatory changes in normal rats on low salt diet [15].
The main nding was that a slight increase of serum uric
acid induced hypertension and afferent arteriole hyper-
trophy associated with glomerular hypertension; addi-
tionally, we found a positive correlation between serum
uric acid and glomerular pressure. This nding suggested
that the vascular lesioninducedby hyperuricemia impairs
the autoregulatory capacity of preglomerular vessels al-
lowing the transmission of systemic hypertension to the
glomerular capillary tuft, resulting in glomerular hyper-
tension [15]. However, these studies were done under
low salt diet, which is a known inductor of renal vasocon-
striction [16], and we were not able to clearly evaluate
the vasoconstrictive effect of the rise of serum uric acid,
as suggested in clinical [8] and experimental [9] reports.
Therefore, in the present study we evaluated the renal
hemodynamic changes associated with the renal vascular
damage induced by mild hyperuricemia in normal rats
under normal salt dietary conditions.
Additionally, there is experimental evidence that re-
lates hyperuricemia with progression of renal damage.
In this regard, Kang et al found that hyperuricemic rem-
nant kidney rats, when compared with control animals,
demonstrated higher blood pressure, greater proteinuria,
higher serum creatinine, greater glomerulosclerosis and
interstitial brosis, and severe vascular disease consisting
of thickening of the preglomerular arteries with smooth
muscle cell proliferation. Allopurinol signicantly re-
duced uric acid levels and blocked the renal functional
and histologic changes [17]. Hence, the second objective
of this study was to evaluate the glomerular microcircula-
tory alterations associated with hyperuricemia in a model
of progressive renal disease induced by subtotal renal
ablation.
METHODS
Experimental design
All animal procedures were approved by the Animal
Care Committee. Male 300 to 350 g Sprague-Dawley rats
were used in all experiments.
Hyperuricemia in normal rats on normal sodium diet
To produce hyperuricemia, rats were administered ox-
onic acid (OA) (Sigma, St. Louis, MO, USA), 750 mg/kg
body weight, by gastric gavage. To prevent the rise of
serumuric acid induced by OA, allopurinol (AP) (Sigma)
was administered in drinking water (150 mg/L). We stud-
ied the following groups: control (N = 8); OA (N = 7);
and OA plus allopurinol (N =9). Systolic blood pressure
and serum uric acid were measured at baseline and after
30 to 33 days of follow-up. Micropuncture studies were
performed at 5 weeks.
Hyperuricemia in remnant kidney (RK) rats on normal
sodium diet
Under light anesthesiawithether, 5/6nephrectomywas
performed by removal of the right kidney and selective
ligation of 2 to 3 branches of left renal artery. To induce
hyperuricemia, rats were givenOA(750 mg/kg) by gastric
gavage starting the day after the renal ablation. To pre-
vent hyperuricemia, APwas administered in the drinking
water (150 mg/L). The following groups were studied: RK
(N=10); RK+OA(N=12); and 5/6 RK+OA+AP(N=
S anchez-Lozada et al: Mild hyperuricemia, cortical vasoconstriction, and glomerular hypertension 239
13). Systolic blood pressure, proteinuria, and serum uric
acid were measured at baseline and after 25 to 28 days
of follow-up. Micropuncture studies were performed at
4 weeks.
Micropuncture
Animals were anesthetized with pentobarbital sodium
(30 mg/kg IP) and placed on a thermoregulated table
to maintain body temperature at 37

C. Trachea, jugular
veins, femoral arteries, and the left ureter were catheter-
ized with polyethylene tubing (PE-240, PE-50, and PE-
10). The left kidney was exposed, placed in a Lucite
holder, sealed with agar, and covered with Ringers solu-
tion. Mean arterial pressure (MAP) was monitored with
a pressure transducer (Model p23 db; Gould, San Juan,
PR) and recorded on a polygraph (Grass Instruments,
Quincy, MA, USA). Blood samples were taken period-
ically and replaced with blood from a donor rat. Rats
were maintained under euvolemic conditions by infu-
sion of 10 mL/kg of body weight of isotonic rat plasma
during surgery, followed by an infusion of 25% polyfruc-
tosan, at 2.2 mL/h (Inutest; Laevosan-Gesellschaft, Linz,
Austria). After 60 minutes, 5 to 6 3-minute collection
samples of proximal tubular uid were obtained to
determine ow rate and polyfructosan concentration.
Intratubular pressure under free-ow and stop-ow con-
ditions and peritubular capillary pressure were measured
in other proximal tubules with a servo-null device (Servo
Nulling Pressure System; Instrumentation for Physiol-
ogy and Medicine, San Diego, CA, USA). Polyfructosan
was measured in plasma samples. Glomerular colloid os-
motic pressure was estimated in protein from blood of
the femoral artery (Ca) and surface efferent arterioles
(Ce). Polyfructosan concentrations were determined by
the technique of Davidson and Sackner [18]. The vol-
ume of uid collected from individual proximal tubules
was estimated from the length of the uid column in a
constant bore capillary tube of known internal diameter.
Concentration of tubular polyfructosan was measured by
the method of Vurek and Pegram [19]. Single nephron
glomerular ltration rate (SNGFR) was calculated using
the formula: SNGFR = (TF/P)
PF
V, where PF is the
concentration of polyfructosan in tubular uid (TF) and
plasma (P), and V is the tubular ow rate which is ob-
tained by timing the collection of tubular uid [20].
Protein concentration in afferent and efferent samples
was determined according to the method of Viets et al
[21]. MAP, GFR, glomerular capillary hydrostatic pres-
sure (PGC), single-nephron plasma ow (QA), afferent
(AR) and efferent (ER) resistances, and Kf were calcu-
lated according to equations given elsewhere [20].
Evaluation
In all studies, systolic blood pressure (SBP) was mea-
sured by tail cuff sphygmomanometer using an auto-
mated system (Narco Biosystems, Houston TX, USA).
All animals were preconditioned for blood pressure
measurements 1 week before each experiment. Protein-
uria was determined by turbidimetry by the method of
trichloroacetic acid[22]. Serumuric acidwas measuredby
colorimetric phosphotungstic acid method using a com-
mercial kit (Wiener Lab, Rosario, Argentina).
Renal histology
After the micropuncture study, kidneys and rem-
nant kidneys were washed by perfusion with phosphate-
buffered saline (PBS) and then xed with 4%
paraformaldehyde. Renal biopsies were embedded in
parafn. Four lm sections of xed tissue were stained
with periodic-acid Schiff (PAS) reagent. Arteriolar mor-
phology was assessed by indirect peroxidase immunos-
taining for alpha-smooth muscle actin (Dako Corp.,
Carpinteria, CA, USA). Renal sections incubated with
normal rabbit serum were used as negative controls for
immunostaining against specic smooth-muscle actin l-
aments. For CD-5 negative control, slides were treated
with nonrelevant antisera. In addition, in remnant kid-
ney rats, glomerulosclerosis and brosis were assessed
with Massons trichrome stain, and tubulointerstitial lym-
phocytes were assessed by indirect immunoperoxidase
immunostaining for anti-CD-5.
Quantication of morphology
Quantications were performed blinded. Afferent ar-
terioles (AA) were identied by their location adjacent
to the vascular pole of the glomerular tuft, the presence
of aninternal elastic lamina, andby having fewer thinat-
tened endothelial cells than the efferent arteriole [23, 24].
We excludedfrommeasurement all vessels showing more
thanone layer of endothelium, as suggestedby Clappet al
[25]. For each arteriole, the outline of the vessel and its
internal lumen (excluding the endothelium) was gener-
ated using computer analysis to calculate the total medial
area (outlineinline), in 10 arterioles per biopsy. The
media/lumen ratio was calculated by the outline/inline
relationship.
Glomerulosclerosis was evaluated in Massons
trichrome stained sections. The degree of sclerosis was
scored as follows: in each biopsy, the number of glomeruli
with segmental, mesangial, and global sclerosis, as well
as normal glomerular tufts, was assessed. Mesangial
sclerosis was dened as a peripheral segmental or
global increase in mesangial matrix with obliteration of
capillary loops, far from the hilus. The resulting index in
each animal was expressed as the percent of sclerosed
glomeruli.
Tubulointerstitial brosis evaluation was performed
by a pathologist unaware of the origin of each kidney
section. Sections were stained with Massons trichrome.
240 S anchez-Lozada et al: Mild hyperuricemia, cortical vasoconstriction, and glomerular hypertension
Ten noncrossed elds of cortex (640 477 mm at 10)
per biopsy were analyzed by light microscopy (Olympus
BX51; Olympus American, Melville, NY, USA) and cap-
tured with a digital video camera (CoolSnap Pro; Me-
dia Cybernetics, Silver Spring, MD, USA). Pictures were
processed on a computer and analyzed using Image-Pro-
Plus 5.0 (Media Cybernetics) and Adobe Photoshop 7
(Adobe Systems, San Jose, CA). Taking advantage of the
capabilities of color recognition of this software, posi-
tive blue color areas were selected and quantied in pixel
units, previous exclusion of glomeruli, and vessels from
the eld. For each examined eld, the number of positive
areas was expressed as a fraction of the tubulointerstitial
area (blue positive areas divided by overall eld area).
Finally, for each biopsy, the mean fractional amount of
positive blue areas was obtained by averaging the values
from 10 examined elds.
Tubulointerstitial lymphocyte inltration was studied
with the immunoperoxidase technique as described be-
fore [26, 27]. Briey, tissues were successively washedand
incubated with 20 mLof ExtrAvidin, 2.5 mg/mL(Sigma),
and with 30 mL0.001%biotin in PBS. Afterwards, tissues
were incubated for 2 hours at 37

C with 50 mL of the
corresponding primary monoclonal antibody (see later)
diluted 1:30 in Tris-saline buffer (TSB), pH 7.8. After
washing in TSB for 15 minutes, tissues were incubated
for 1 hour with 30 mL of rat antimouse IgG (ab) biotin-
conjugated fragments with minimal cross-reactivity with
human, horse, and rat serum proteins (Accurate Chemi-
cal Corp, Westbury, NY, USA), and nally for 30 minutes
with 60 mL of peroxidase-conjugated ExtrAvidin. After
a nal wash, tissues were incubated for 15 minutes in di-
aminobenzidine and H
2
O
2
in TSB.
The primary antibody used was anti CD5 (mouse mon-
oclonal anti rat thymocytes and lymphocytes) (Biosource
International, Camarillo, CA). Results were expressed as
positive cells per mm
2
.
Statistical analysis
Values are expressed as mean standard error (SE).
Differences between groups were evaluated by analy-
sis of variance (ANOVA) with appropriate correction
for multiple comparisons (Bonferroni). The relation be-
tween variables was assessed by correlation analysis.
RESULTS
Hyperuricemia in normal rats on a normal sodium diet
General characteristics of the model. As previously de-
scribed, under low salt dietary conditions, oxonic acid
administration induced a 2-fold increment of serum uric
acid in association with a slight, but signicant, increment
of systolic blood pressure demonstrated by intra-arterial
and tail-cuff measurements [15] (Table 1). Concurrent
administration of allopurinol prevented the increase of
T
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S anchez-Lozada et al: Mild hyperuricemia, cortical vasoconstriction, and glomerular hypertension 241
r = 0.47
P = 0.03
75
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,

n
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Serum uric acid, mg/dL
r = 0.73
P = 0.0002
A
Fig. 1. Effect of afferent arteriole thickening induced by hyperuricemia on hemodynamic parameters in normal rats. Values of serum uric acid pos-
itively correlated with AA media/lumen (A). Arteriolopathy was associated with glomerular pressure (B). Thickening of AA negatively correlated
with SNGFR (C) and positively correlated with afferent resistance (D). , control; , oxonic acid; , OA+ allopurinol.
serum uric acid, as well as the hypertension (Table 1). In
addition, individual values of serumuric acid and systolic
blood pressure correlated positively (P < 0.05). Values
of body weight were similar among the studied groups
(Table 1).
Micropuncture studies. Results of glomerular hemo-
dynamic studies are summarized in Table 1. There were
nochanges inGFRamong groups. Hyperuricemia was as-
sociated with cortical vasoconstriction, as evidenced by
a 35% decrease of SNGFR and negative correlation be-
tween individual values of uric acid and SNGFR (r =
0.6, P = 0.004). The decrease in SNGFR resulted from
lower QA and Kf despite the counterbalancing effect of
the signicant increment of PGC. In addition, afferent
and efferent resistances were 45% and 64% higher, re-
spectively. Concomitant treatment with allopurinol pre-
vented these alterations; PGC and SNGFR remained
unchanged, and glomerular plasma ow increased to
higher values than OA treated and control rats. Incre-
ment in QA was due to numerically lower values of AR
and a signicant decrease of ER. Additionally, we found
a negative correlation between serum uric acid and Kf
(r = 0.5, P = 0.02), and positive correlations of serum
uric acid with PGC (r = 0.7, P = 0.0002), AR (r = 0.5,
P = 0.007) and ER (r = 0.7, P < 0.0001).
Histologic examination. As previously reportedinlow
salt dietary conditions, oxonic acid administration in-
duced hypertrophy of afferent arteriole as disclosed by
a signicant increment of media to lumen ratio com-
pared to control rats [15] (Table 1, Fig. 3). Allopuri-
nol treatment prevented thickening of afferent arterioles
(Table 1). We found positive correlations between indi-
vidual values of serumuric acid and media to lumen ratio
(r = 0.73, P = 0.0002); in addition, arteriolopathy (M/L
ratio) correlated positively with glomerular pressure
(r = 0.75, P < 0.0001) and afferent resistance (r = 0.54,
P=0.01). Finally a negative correlationbetweenM/Land
SNGFR was demonstrated (r =0.47, P =0.03) (Fig. 1).
Hyperuricemia in remnant kidney rats
General characteristics of the model. Renal ablation
was characterized by marked hypertension, proteinuria,
andnormal values of serumuric acid4weeks after surgery
(Table 2). Administration of oxonic acid induced a 2-fold
increaseof serumuric acid(P<0.001) that was associated
242 S anchez-Lozada et al: Mild hyperuricemia, cortical vasoconstriction, and glomerular hypertension
T
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with further increase of 30 mmHg of SBP (P<0.05), and
a 2-foldadditional rise of proteinuria (P<0.01) (Table 2).
Concomitant administration of allopurinol prevented hy-
peruricemia (P < 0.001) and proteinuria (P < 0.01), but
had only a marginal effect on SBP (P = NS) (Table 2).
There were no differences of body weight among studied
groups (Table 2).
Micropuncture studies. The results obtained in the
micropuncture studies are summarized in Table 2. Al-
though whole kidney GFRwas similarly reduced by renal
ablation in all groups (table 2), measurement of sin-
gle nephron function demonstrated marked differences
in glomerular adaptation between RK control rats and
those receiving OAandOA+AP. RKcontrol rats showed
the typical hyperltration as has been reported in other
studies. In contrast, SNGFR in OA treated was 40%
lower than the RK control group (40.6 vs. 65 nL/min, P <
0.01). Treatment with AP partially prevented the change
of SNGFR (53 nL/min, P = NS).
The reduction of SNGFR in OA treated animals re-
sulted from reduction of 2 of the determinants of GFR,
the glomerular plasma ow and the ultraltration coef-
cient Kf, while glomerular capillary pressure persisted
with similar elevation as the RK control group (62 vs.
61 mmHg, P=NS). In the OAgroup, glomerular plasma
ow was 40% and Kf 50% lower than RK control group
(GPF: 147 vs. 243 nL/min, P < 0.01; Kf: 0.02 vs. 0.04 nL/
s/mm Hg, P < 0.05). Glomerular underperfusion in hy-
peruricemic rats was the result of a 120% higher afferent
resistance and 80% higher efferent resistance compared
to RK control animals (AR: 4.6 vs. 2.1 dyn/s/cm
5
, P <
0.01; ER: 1.8 vs. 1 dyn/s/cm
5
, P < 0.01). Thus, despite
a proportional higher increase in afferent than efferent
resistances in OA group, glomerular pressure remained
elevateddue tothe simultaneous rise insystolic andmean
arterial pressure. Treatment with AP partially prevented
the cortical ischemia (Table 2); QAand SNGFRwere nu-
merically higher than hyperuricemic rats. However, the
most striking effect of APwas the prevention of glomeru-
lar hypertension in the presence of systemic hyperten-
sion. Infact, PGCwas normal andsignicantly lower than
RK hyperuricemic rats (P <0.001), as well as lower than
RK control (P < 0.01), indicating that allopurinol treat-
ment restored autoregulatory capacity of preglomerular
arteriole to prevent increased transmission of pressure to
the glomerular capillary.
Histologic examination. Results of histologic exami-
nation are summarized in Table 3. Analysis of afferent ar-
teriole morphology by immunostaining for alpha-smooth
muscle actin demonstrated that extensive renal mass re-
duction induced thickening of the afferent arteriole wall
evaluated by the media to lumen ratio, as we previously
reported [27]. Oxonic acid administration considerably
aggravated hypertrophy of preglomerular vessels as in-
dicated by higher media to lumen ratio than control (6.4
S anchez-Lozada et al: Mild hyperuricemia, cortical vasoconstriction, and glomerular hypertension 243
Table 3. Histologic ndings in remnant kidney rats
TI brosis
Groups AA M/L arbitrary units Glomerulosclerosis % TI CD5 + cells/mm
2
RK 5.0 0.39 733.3 156.3 24 10 27.8 2.13
OA 6.4 0.37 780.2 55.0 33 7 26.1 2.99
OA+AP 1.8 0.31 676.9 107.3 27 4 15.8 0.80
RK vs. OA <0.05 NS NS NS
OA vs. OA+AP <0.001 NS NS <0.05
RK vs. OA+AP <0.001 NS NS <0.01
10.0
7.5
5.0
2.5
0.0
A
A

m
e
d
i
a
/
l
u
m
e
n
0 10 20 30 40 50
TI CD5+, cells/mm
2
r = 0.6
P = 0.006
C
1500
1000
500
0
F
i
b
r
o
s
i
s
,

a
r
b
i
t
r
a
r
y

u
n
i
t
s
40 50 60 70 80
Glomerular pressure, mm Hg
r = 0.54
P = 0.01
D
0.0 2.5 5.0 7.5 10.0
AA media/lumen
80
70
60
50
40
G
l
o
m
e
r
u
l
a
r

p
r
e
s
s
u
r
e
,

m
m

H
g
r = 0.49
P = 0.02
B
10.0
7.5
5.0
2.5
0.0
A
A

m
e
d
i
a
/
l
u
m
e
n
0.0 2.5 5.0 7.5 10.0
Serum uric acid, mg/dL
r = 0.47
P = 0.02
A
Fig. 2. Effect of afferent arteriole thickening induced by hyperuricemia in RK rats. Values of serum uric acid positively correlated with AA
media/lumen(A). Arteriolopathy was associatedwithglomerular pressure (B). Tubulointerstitial inltrationof CD5+cells (lymphocytes) correlated
with AA thickening (C). Glomerular pressure correlated with tubulointerstitial brosis (D). , control RK; , oxonic acid; , OA+ allopurinol.
vs. 4.9, P < 0.05). Allopurinol prevented arteriolopathy
in hyperuricemics to a greater extent than that observed
in RK control rats (AP = 1.8, P vs. Nx < 0.001; P vs. OA
< 0.001). Moreover, media to lumen values in OA+AP
were similar to values found in the present study in nor-
mal rats (Table 1) using the same methodof perfusionand
xation. This suggests that allopurinol exerted an addi-
tional favorable effect besides preventing hyperuricemia
(Fig. 3).
Neither glomerulosclerosis nor tubulointerstitial bro-
sis was different among studied groups. Nevertheless,
tubulointerstitial inltration of CD5-positive cells was
signicantly lower in allopurinol treated rats with respect
to RK control and RK hyperuricemic rats (Nx = 27.8;
OA = 26.1; OA+AP = 15.8; Nx vs. OA+AP, P < 0.01;
OA vs. OA+AP, P < 0.05). No immunostaining was ob-
served when antibodies were omitted or replaced by non-
immune rabbit serum or nonrelevant antisera.
Finally we foundpositive correlations betweenindivid-
ual values of serum uric acid versus media to lumen ratio
(r = 0.5, P = 0.02), M/L versus PGC (r = 0.5, P = 0.02)
and CD5 (r = 0.6, P = 0.006), and PGC versus brosis
(r = 0.5, P = 0.02) and CD5 (r = 0.5, P = 0.01) (Fig. 2).
DISCUSSION
We studied the effect of hyperuricemia-induced pre-
glomerular arteriolopathy on glomerular hemodynamics
in normal and in remnant kidney rats on normal salt diet.
244 S anchez-Lozada et al: Mild hyperuricemia, cortical vasoconstriction, and glomerular hypertension
Fig. 3. Representative microphotographs of afferent arteriole morphology. PAS stain and alpha-actin smooth muscle antibody (1000). Normal
rats on normal salt diet (A), oxonic acid (C), OA+ allopurinol (E); control (RK) (B), RK + oxonic acid (D), RK + OA+ allopurinol (F).
The main nding was that the vascular lesion was asso-
ciated with cortical vasoconstriction and glomerular hy-
pertension in both conditions.
Previously we studied the effect of mild hyperuricemia
in normal rats under low salt diet and obtained similar
results [15], but we were unable to clearly demonstrate
the vasoconstrictive effect suggested in clinical [8] and
experimental [9] studies. In addition to differences in salt
intake, other differences between both studies should be
mentioned. In order to give rats more accurate doses of
OA and achieve better inhibition of uricase, in this work
we administered oxonic acid by daily gavage instead of
a rat chow supplemented with 2% OA. Control values
of SUA were higher in the present study than values re-
ported in the previous studies under conditions of salt
restriction [15]. Despite these differences, oxonic acid
administration did signicantly increase serum uric acid,
and allopurinol prevented this increment. Moreover, be-
cause all animals were maintained in the same conditions,
comparisons remain valid.
S anchez-Lozada et al: Mild hyperuricemia, cortical vasoconstriction, and glomerular hypertension 245
In normal rats, by eliminating RAS activation induced
by low salt diet we were able to reveal the renal vaso-
constrictive effect of hyperuricemia. In fact, SNGFR was
35% lower than controls, and we found a negative cor-
relation between individual values of SUA and SNGFR
and KF, as well as a positive correlation between afferent
and efferent resistances. These ndings are in agreement
with previous experimental and clinical studies, in which
a positive correlation between SUA and renal vascular
resistance was observed. Additionally, hyperuricemia in-
duced glomerular hypertension, as we found in our pre-
vious report [15].
In this study, we conrmed that hyperuricemia pro-
duced arteriolopathy of preglomerular vessels, as in-
dicated by the positive correlation between individual
values of serum uric acid and media to lumen ratio. Fur-
thermore, rats on normal salt intake had 10 mm Hg less
SBP than animals on LSD, and still had similar values
of media to lumen ratio. There is evidence that uric acid
directly stimulates vascular smooth muscle cell prolifera-
tion, whichmay be the mechanismfor the development of
thevascular disease. Raoet al reportedthat uric acidstim-
ulates PDGF-A chain expression and mediates cell pro-
liferation in cultured vascular smooth muscle cells [28].
Consistent with this report, Kang et al [17] found de novo
expression of COX-2 mRNA and proliferation after in-
cubation of VSMC with uric acid. A COX-2 inhibitor or
a TXA2 receptor inhibitor prevented the proliferation in
response to uric acid. In vivo COX-2 was also shown to
be expressed de novo in the preglomerular vessels, and
its expression correlated both with the uric acid levels
and with the degree of smooth muscle cell proliferation
[17]. Our nding agrees with these studies, which demon-
strated that uric acid induces arteriolar hypertrophy by a
mechanism independent of the increment of blood pres-
sure. Moreover, inthe present study the deleterious effect
of arteriolopathy on glomerular hemodynamics was ev-
idenced by the positive correlations between media to
lumen ratio and glomerular pressure, and afferent re-
sistance and negative correlation between M/L versus
SNGFR.
These results suggest that in normal rats a mild incre-
ment of serum uric acid induces preglomerular arteri-
olopathy that results in transmission of systemic hyper-
tension to glomerular capillary tuft, which, in concert
withhigher efferent resistanceinducedbyhyperuricemia,
further enhances glomerular hypertension. In addition,
thickening of the afferent arteriole wall induces higher af-
ferent resistance, and the reduction of glomerular plasma
owand Kf result in lower single nephron GFR. Systemic
hypertension could result from the consequence of the
decrement in ltered load, or as a consequence of renal
ischemia resulting from the decrease in renal blood ow.
Previous studies by Kang et al showed that hyper-
uricemia canaccelerate progressionof renal damage [17].
In that study, in rats with subtotal renal ablation induced
by polectomy, mildincrement of serumuric acidwas asso-
ciated with worse renal function, greater structural dam-
age, and severe vascular damage. Because we have shown
that arteriolopathy of afferent arteriole in normal [15]
and 5/6 nephrectomy rats [27] is associated with alter-
ations in glomerular microcirculation that contribute to
perpetuate progression of renal damage, in the present
study we evaluated glomerular hemodynamics changes
caused by the vascular lesion induced by hyperuricemia
in rats with 5/6 nephrectomy produced by ligation of sev-
eral branches of the renal artery. Control RK rats devel-
oped severe hypertension and proteinuria, as has been
reported in this model of renal ablation [27, 29]. Both
parameters increased further with hyperuricemia, allop-
urinol restoredproteinuria, andtendedto, but didnot sig-
nicantly reduce the blood pressure. The main nding of
these studies was that in remnant kidney rats, mild hyper-
uricemia produced profound renal cortical vasoconstric-
tion. Single nephron glomerular plasma ow and GFR
fell by 40%, ultraltration coefcient Kf also decreased
in the same proportion. There was a higher proportional
increase in afferent resistance than efferent resistance.
However, despite a more intense preglomerular vasocon-
striction due to arteriolopathy, glomerular hypertension
remained unchanged. This apparent paradox can be ex-
plained by the concomitant further elevation of arterial
pressure in hyperuricemic rats. Glomerular pressure is
determined by the relative changes of afferent and ef-
ferent resistances, as well as by the changes in arterial
pressure. Increased glomerular pressure can occur in the
presence of a relatively greater increase of afferent than
efferent resistance, if at the same time there is an incre-
ment of arterial pressure of sufcient magnitude. This was
previously observedinagedSHRrats [30], inrats with5/6
nephrectomy when compared with normal controls [29],
and in Fawn Hooded rats after NO synthesis inhibition
with L-NAME [31].
As we showed previously, morphometric analysis of af-
ferent arteriole in control RKrats displayed hypertrophy
of the vascular wall [27]. However, the rise in serum uric
acid markedly accentuated the arteriolopathy by thick-
ening the arteriolar wall and increasing the media to lu-
men ratio by 30%. Allopurinol treatment partly restored
the functional changes; however, it fully prevented arte-
riolopathy. Arteriolar wall thickness was normal and me-
dia to lumen ratio signicantly lower than hyperuricemic
and control RK groups. Interestingly, preservation of
vascular structure was associated with maintaining nor-
mal glomerular pressure, despite systemic hypertension,
which denotes a normal autoregulatory response of pre-
glomerular vessels to the rise of arterial pressure. The
contribution of preglomerular vascular lesion in deter-
mining the elevation of glomerular capillary pressure was
further evidenced by a positive signicant correlation of
246 S anchez-Lozada et al: Mild hyperuricemia, cortical vasoconstriction, and glomerular hypertension
individual values of media to lumen ratio and glomeru-
lar pressure. In addition, a signicant positive correlation
was found between individual values of serum uric acid
and media to lumen ratio, underscoring the role of uric
acid in stimulating proliferation of vascular smooth mus-
cle cells.
Recently we reported that arteriolopathy of affer-
ent arteriole by perpetuating glomerular hemodynamic
stress contributes to progression of renal disease [27]. In
renal ablated rats, histologic examination and morphom-
etry showed thickening of afferent arterioles as disclosed
by a signicant increase in media/lumen ratio indicating
hypertrophy of the vessel wall. Proliferation of vascular
smooth muscle cells and increased collagen deposition
might be expected to increase rigidity of the vascular
wall and, thus, limit its capacity to contract in response to
higher perfusion pressure [32]. Maneuvers that prevent
the accompanying inammatory process, like treatment
with the heparinoid pentosan polysulfate [29] and the
immunosuppressive drug, mofetil mycophenolate [27],
preserved arteriolar structure, maintaining normal
glomerular pressure despite persisting systemic hyper-
tension indicating a normal autoregulatory response. In
the present study, allopurinol treatment reduced tubu-
lointerstitial inltration of CD5+ cells compared to OA
treated and RKcontrol rats. This effect could be partially
responsible of the fully prevention of preglomerular ar-
teriolopathy and glomerular hypertension in allopurinol
treated group, besides its lowering uric acid effect. Al-
though after 30 days of follow-up we were not able to
detect a signicant increase in tubulointerstitial injury,
glomerulosclerosis, or CD5+ cells inltration in hyper-
uricemic RK rats compared to controls, probably a more
prolonged follow-up would uncover the effect of renal
vasoconstriction on these parameters. In fact, in the study
of Kang et al [17], in which control and hyperuricemic 5/6
nephrectomy rats were followed for 6 weeks, they found
signicantly more glomerulosclerosis and tubulointersti-
tial brosis at that time point. In addition, Nakagawa
et al followed rats with oxonic acidinduced mild hy-
peruricemia for 6 months [33], and reported that they
eventually developed glomerular hypertrophy and
glomerulosclerosis with albuminuria.
On the other hand, hypertrophy of vascular smooth
muscle cells and expanded ECM on vascular wall may
critically reduce the lumen of preglomerular vessels, in-
ducing the decline of blood ow to glomeruli and post-
glomerular ischemia, which is a well-known stimulus to
produce tubulointerstitial brosis and salt-sensitive hy-
pertension [34]. In the present study, glomerulosclerosis
andbrosis weresimilar amongstudiedgroups. However,
the correlation between individual values of glomerular
pressure and brosis suggests that transmission of sys-
temic hypertension to peritubular capillaries may dam-
age them, further incrementing ischemia and brosis. In
addition to these hemodynamic effects, experimental hy-
peruricemia has also been associated with endothelial
dysfunction and lower serum levels of nitrates/nitrites,
indicating decreased synthesis of nitric oxide [abstract;
Finch J et al, J Am Soc Nephrol 14:143A, 2004]. In this
regard, it was shown that inhibition of NO production
markedly accelerates progression of renal damage inde-
pendently of systemic hypertension and in association
with loss of microvascular endothelium [35]. Bohle et al
also suggested a critical role for the peritubular capillar-
ies in the progressive interstitial brosis in human renal
disease [36].
CONCLUSION
Renal arteriolopathy induced by mild hyperuricemia
results in an impaired capacity of preglomerular vessels
tomaintainconstancy of glomerular pressure inthe phase
of arterial hypertension, which results in glomerular hy-
pertension. In addition, lumen obliteration induced by
vascular wall thickening results in severe vasoconstric-
tion, decreasing renal plasma ow, GFR, and perfusion
to peritubular capillaries. The resulting ischemia is a po-
tent stimulus that induces tubulointerstitial inammation
and brosis, as well as arterial hypertension. Evidence
for these alterations is provided by studies in which mild
hyperuricemia was responsible for later development of
salt-sensitive hypertension in normal rats [37]. Further-
more, it is possible that as the arteriolar changes become
even more severe, glomerular ischemia and collapse with
a decrease in glomerular pressure could take place, as
has been postulated to occur with prolonged and severe
hypertension [38]. Thus, these studies provide a potential
mechanism by which hyperuricemia can mediate hyper-
tension and renal disease.
ABBREVIATIONS
AA Afferent arteriole
AP Allopurinol
AR Afferent resistance
CD5 Rat thymocytes and lymphocytes antigen
ER Efferent resistance
GFR Glomerular ltration rate
Kf Ultraltration coefcient
M/L Media to lumen ratio
MAP Mean arterial pressure
OA Oxonic acid
PGC Glomerular capillary hydrostatic pressure
QA Single-nephron plasma ow
RK Remnant kidney
SBP Systolic blood pressure
SNGFR Single nephron GFR
ACKNOWLEDGEMENTS
This paper was supportedinpart by grant 37275-Mfromthe Mexican
Council of Science and Technology (CONACYT) and NIH grants HL-
68607 (to RJJ and JH-A), DK-52121 (RJJ) and DK-64233 (RJ). Part of
this work was presented at the 36th Annual Meeting of the American
S anchez-Lozada et al: Mild hyperuricemia, cortical vasoconstriction, and glomerular hypertension 247
Society of Nephrology, November 1317, 2003, San Diego, California.
We thank Benito Ch avez Renter a for technical assistance in histologic
processing.
Reprint requests to Laura G. S anchez-Lozada, Ph.D., Dept. Nephrol-
ogy, INC Ignacio Chavez, Juan Badiano 1, 14080-Mexico City, Mexico.
E-mail: lgsanchezlozada@hotmail.com
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