1. health and fitness
2. disease

J Am Soc Nephrol 13: 2898–2908, 2002
How To Fully Protect the Kidney in a Severe Model of
Progressive Nephropathy: A Multidrug Approach
CARLA ZOJA,* DANIELA CORNA,* DAVIDE CAMOZZI,* DARIO CATTANEO,*
DANIELA ROTTOLI,* CRISTIAN BATANI,* CRISTINA ZANCHI,*
MAURO ABBATE,* and GIUSEPPE REMUZZI*†
*Mario Negri Institute for Pharmacological Research, Bergamo, Italy; and †Unit of Nephrology and Dialysis,
Azienda Ospedaliera, Ospedali Riuniti di Bergamo, Bergamo, Italy.
Abstract. The current therapy for chronic proteinuric nephropathies is angiotensin-converting enzyme inhibitors (ACEi),
which slow, but may not halt, the progression of disease, and
which may be not effective to the same degree in all patients.
In accelerated passive Heymann nephritis (PHN), this study
assessed the effect of combining ACEi with angiotensin II
receptor antagonist (AIIRA) and with statin that, besides lowering cholesterol, influences inflammatory and fibrogenic processes. Uninephrectomized PHN rats were divided into four
groups (n ⫽ 10 each) and daily given oral doses of the
following: vehicle; 40 mg/L lisinopril; 100 mg/L lisinopril plus
L-158,809; 0.3 mg/kg lisinopril plus L-158,809 plus cerivastatin. Treatments started at 2 mo when rats had massive proteinuria and signs of renal injury and lasted until 10 mo.
Increases in BP were equally lowered by treatments. ACEi
kept proteinuria at levels comparable to pretreatment and numerically lower than vehicle. The addition of AIIRA to lisin-
opril was more effective, being proteinuria reduced below
pretreatment values and significantly lower than vehicle. When
cerivastatin was added on top of ACE inhibition and AIIR
blockade, urinary protein regressed to normal values and renal
failure was prevented. Renal ACE activity was increased threefold in PHN, it was inhibited by more than 60% after ACEi,
and decreased below control values with triple therapy. Cerivastatin inhibited ACE activity by 30%. Glomerulosclerosis,
tubular damage and interstitial inflammation were ameliorated
by ACEi alone or combined with AIIRA, and prevented by
addition of statin. TGF-␤1 mRNA upregulation in PHN kidney
was partially reduced after ACEi or combined with AIIRA and
almost normalized after adding statin. Cerivastatin inhibited
TGF-␤1 gene upregulation by 25%. These data suggest a
possible future strategy to induce remission of proteinuria,
lessen renal injury, and protect from loss of function in those
patients who do not fully respond to ACEi therapy.
Proteinuria is a major determinant of progression in both
experimental and human nephropathies. High levels of urinary
proteins, which reflect excess protein trafficking through the
glomerulus, are associated with a faster course of disease (1,2).
Experimental observations suggested mechanisms whereby enhanced tubular reabsorption of proteins contributes substantially to promote interstitial inflammatory and fibrogenic reactions that evolve to renal scarring. Overloading of proximal
tubular cells in culture with plasma proteins enhanced the
production of proinflammatory substances such as endothelin-1, monocyte chemoattractant protein-1 (MCP-1), and RANTES (3–5). Transforming growth factor ␤ (TGF-␤) was also
upregulated in proximal tubular cells on protein challenge (6).
Mediators were released preferentially into the basolateral cell
medium in a fashion that in the kidney would incite interstitial
inflammation and fibrosis.
Angiotensin (Ang) converting enzyme inhibitors (ACEi),
which reduce protein trafficking and its long-term toxicity,
offer superior protection against renal damage. In virtually all
experimental models of chronic proteinuric nephropathy, ACEi
limit proteinuria and renal injury when treatment starts soon
after insult (7–9). By contrast, a delayed administration may
not be sufficient to reduce proteinuria and to slow the progression of the disease (10,11). Thus in diabetic rats, ACEi normalized proteinuria and protected against renal structural
changes when treatment was started early in the course of the
disease (23 wk), but not at the time when proteinuria was
higher (32 wk) (12). In the accelerated model of passive
Heymann nephritis (PHN), lisinopril limited proteinuria and
renal injury if given since 7 d after disease induction, whereas
it failed, even at a very high dose, if given since 4 mo (10,13).
Similar considerations apply to the clinical setting (14). In
proteinuric patients, ACEi slow, but do not invariably halt,
progressive nephropathy (15,16). Thus, treatments that synergize with ACEi in further limiting proteinuria and/or limiting
interstitial injury have been proposed. The combination of
ACEi and AngII type 1 receptor antagonist (AIIRA) has been
suggested as a way to maximize renin angiotensin system
(RAS) blockade at different levels: reduction of AII availability for binding to angiotensin type I (AT1) receptor and direct
inhibition of AII binding to AT1 (17,18). The rationale for the
Received May 22, 2002. Accepted August 2, 2002.
Correspondence to Dr. Carla Zoja, ‘Mario Negri’ Institute for Pharmacological
Research, Via Gavazzeni, 11, 24125 Bergamo, Italy. Phone: 39-0-35-319-888;
Fax: 39-035-319-331; E-mail: zoja@marionegri.it
1046-6673/1312-2898
Journal of the American Society of Nephrology
Copyright © 2002 by the American Society of Nephrology
DOI: 10.1097/01.ASN.0000034912.55186.EC
J Am Soc Nephrol 13: 2898–2908, 2002
Angiotensin II Blockade Plus Statin Prevent Progressive Nephropathy
combination therapy rests on the evidence that long-term ACEi
treatment results in the accumulation of AngI, which may
escape ACE inhibition and generate AII that through AT1
receptor causes deleterious effects to the kidney, such as vasoconstriction, inflammation, and fibrosis (19,20). Studies
have shown in fact that in the presence of ACE inhibition, AII
may be produced by alternative pathways, including chymase
(17). Angiotensin receptor blockers could overcome these
shortcomings of ACEi by directly antagonizing the AT1 receptor. In addition, the blockade of AT1 receptor in the presence of elevated AII levels can result in the stimulation of the
angiotensin subtype 2 (AT2) receptor, which seems to counteract the vasoconstrictor and proliferative action of AT1 (17).
Of interest is the evidence that combining an ACE inhibitor
and an AIIRA reduced plasma and kidney AII levels more than
these agents did alone, providing a potential mechanism for
their synergism in reducing proteinuria and BP (21).
So far, experimental and clinical studies on combination
therapy with ACEi and AIIRA are few and the results are
conflicting. In diabetic transgenic (mRen-2)27 rats, low-dose
perindopril plus valsartan gave more benefit on the kidney
versus monotherapy (22). In rats with renal mass reduction,
ACEi plus AIIRA resulted in greater renal protection (23), but
not in a study when the doses were adjusted to maintain BP
control comparable to single drugs (24). In rats with adriamycin nephrosis, addition of AIIRA failed to overcome resistance
to ACE inhibition (11). In a small study in patients with IgA
nephropathy, ACEi plus AIIRA was at least additive in decreasing proteinuria, in contrast to no effect when the dose of
either drug was doubled (25). Among type 2 diabetic patients
with microalbuminuria, the combined drugs also afforded
greater reductions in BP and albuminuria (26). In a study of 23
patients with nondiabetic chronic nephropathies at comparable
BP control, combined therapy with halved doses of ACEi and
AIIRA decreased proteinuria better than full doses alone (27).
However, AIIRA added on top of maximal ACE inhibition was
not superior to ACEi alone in decreasing proteinuria in 16
patients with various chronic renal diseases (28).
Statins have pleiotropic properties that complement their
cholesterol-lowering effects and may provide additional benefit in combination therapy. By interfering with prenylation of
Ras and Rho family small GTP-binding proteins, they block
the activation of mitogen-activated protein kinase signaling
pathways and transcription factors including NF-␬B and AP-1
(29 –31), which regulate the expression of inflammatory, vasoactive, and fibrogenic genes critical to renal disease progression. Combining ACEi with a statin had more renal protective
effect than single therapy in rats with puromycin-induced nephrotic syndrome (32) as well as in rats subjected to 5/6
nephrectomy (33). We have recently documented that in severe
passive Heymann nephritis (PHN) resistant to ACEi alone,
combination of lisinopril with simvastatin given from month 4
to 10 of disease prevented proteinuria from worsening and also
limited tubulointerstitial damage (10).
In the present study, we assessed whether a multidrug approach with ACEi, AIIRA, and statin could even reverse proteinuria and renal disease progression in rats with accelerated
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PHN treated in the phase of overt proteinuria. Renal TGF-␤1
expression was also evaluated.
Materials and Methods
Experimental Design
Male Sprague-Dawley rats (Charles River Italia s.p.a., Calco, Italy)
with initial body weights of 300 to 350 g were used in this study.
Animal care and treatment were conducted in accordance with the
institutional guidelines that are in compliance with national (Decreto
Legislativo n.116, Gazzetta Ufficiale suppl 40, 18 febbraio 1992,
Circolare n.8, Gazzetta Ufficiale 14 luglio 1994) and international
laws and policies (EEC Council Directive 86/609, OJL358 –1, December 1987; Guide for the Care and Use of Laboratory Animals,
U.S. National Research Council, 1996). All animals were housed in a
room in which the temperature was kept constant on a 12-h dark/12-h
light cycle and allowed free access to standard diet containing 20%
protein by weight and tap water. Passive Heymann nephritis (PHN)
was induced in non-anesthetized rats by a single intravenous injection
of 0.4 ml/100 g body wt of rabbit anti-Fx1A antibody. Unilateral
nephrectomy at day 7, when animals were proteinuric, was performed
to accelerate the onset of renal histologic damage (34). Two months
later, rats were divided into five groups and daily treated up to 10 mo
as follows: vehicle (group 1, n ⫽ 10); 40 mg/L lisinopril (AstraZeneca; Basiglio, Milan, Italy) in the drinking water (group 2, n ⫽ 10); 40
mg/L lisinopril plus 100 mg/L L-158,809 (Merck & Co., Inc., Rahway, NJ) (group 3, n ⫽ 10); 40 mg/L lisinopril plus 100 mg/L
L-158,809 plus 0.3 mg/kg cerivastatin (group 4, n ⫽ 10); 0.3 mg/kg
cerivastatin (Bayer AG, Wuppertal, Germany) (group 5, n ⫽ 10).
Doses of lisinopril, L-158,809, and cerivastatin were chosen on the
basis of previously published studies (8,35). A group of normal rats
followed up to 10 mo served as control (group 6, n ⫽ 6). In addition,
five PHN rats together with four age-matched normal rats were
sacrified 2 mo after disease induction for renal histologic evaluation.
Systolic BP and urinary protein excretion were measured every 2 mo.
Serum creatinine was evaluated at baseline and at months 2 (before
treatment), 4, 8, and 10. Serum levels of cholesterol, triglycerides,
aspartate transaminase (AST), and alanine aminotransferase (ALT)
were assessed at the end of the study. At month 10, rats were
anesthetized and kidneys were removed for measurement of ACE
activity, histology and immunohistochemistry, and total RNA preparation to assess TGF-␤1 mRNA by Northern blot analysis.
Systolic BP (SBP) was recorded in conscious rats by tail plethysmography (IITC Life Science, Woodland Hills, CA). Twenty-four–
hour urine samples were collected using metabolic cages, and proteinuria was determined by modified Coomassie blue G dye-binding
assay for proteins with BSA as standard. Blood was collected from the
tail vein of anesthetized animals. Serum was obtained after whole
blood clotting and kept frozen at ⫺20°C until assayed. Creatinine was
measured by alkaline picrate method. Serum cholesterol, triglycerides,
and transaminase levels were measured using an autoanalyzer (CX5,
Beckman Instruments Inc., Fullerton, CA).
Measurement of ACE Activity
Renal tissue was homogenized in distilled water and centrifuged at
12,000 ⫻ g for 10 min at 4°C. The resulting supernatant was used for
ACE activity determination by a spectrophotometric method (Sigma).
ACE activity was expressed as relative units per milligram protein of
tissue.
Renal Histology
The removed kidneys were fixed for 6 h in Dubosq-Brazil, dehydrated in alcohol, and embedded in paraffin. Kidney samples were
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Journal of the American Society of Nephrology
J Am Soc Nephrol 13: 2898–2908, 2002
sectioned at 3-␮m intervals, and the sections were stained with Masson’s trichrome, hematoxylin and eosin, and periodic acid-Schiff
reagent (PAS stain). Tubular (atrophy, casts, and dilatation) and
interstitial changes (fibrosis and inflammation) were graded from 0 to
4⫹ (0, no changes; 1⫹, changes affecting ⬍25% of the sample; 2⫹,
changes affecting 25 to 50% of the sample; 3⫹, changes affecting 50
to 75% of the sample; 4⫹, changes affecting 75 to 100% of the
sample). At least 100 glomeruli were examined for each animal, and
the extent of glomerular damage was expressed as the percentage of
glomeruli presenting sclerotic lesions. All renal biopsies were analyzed by the same pathologist who was unaware of the nature of the
experimental groups.
Immunohistochemical Analyses
Mouse monoclonal antibodies were used for the immunohistochemical detection of ED-1 antigen present in rat monocytes and
Table 1. Time course of body weight (g) in PHN ratsa
Groups
PHN ⫹ vehicle
PHN ⫹ ACEi
PHN ⫹ ACEi ⫹ AIIRA
PHN ⫹ ACEi ⫹ AIIRA ⫹ cerivastatin
PHN ⫹ cerivastatin
Control
0 mo
2 mo
(before
treatment)
4 mo
6 mo
8 mo
10 mo
305 ⫾ 5
(n ⫽ 10)
315 ⫾ 3
(n ⫽ 10)
323 ⫾ 10
(n ⫽ 10)
307 ⫾ 6
(n ⫽ 10)
304 ⫾ 4
(n ⫽ 10)
324 ⫾ 7
(n ⫽ 6)
532 ⫾ 12
(n ⫽ 10)
538 ⫾ 15
(n ⫽ 10)
506 ⫾ 17
(n ⫽ 10)
519 ⫾ 13
(n ⫽ 10)
542 ⫾ 23
(n ⫽ 10)
562 ⫾ 23
(n ⫽ 6)
598 ⫾ 14
(n ⫽ 10)
592 ⫾ 17
(n ⫽ 10)
538 ⫾ 24b
(n ⫽ 10)
526 ⫾ 24bdf
(n ⫽ 8)
589 ⫾ 25
(n ⫽ 10)
631 ⫾ 22
(n ⫽ 6)
634 ⫾ 15
(n ⫽ 10)
626 ⫾ 21
(n ⫽ 10)
571 ⫾ 31b
(n ⫽ 10)
569 ⫾ 21ce
(n ⫽ 7)
623 ⫾ 27
(n ⫽ 9)
682 ⫾ 23
(n ⫽ 6)
657 ⫾ 17
(n ⫽ 9)
636 ⫾ 25
(n ⫽ 10)
603 ⫾ 35
(n ⫽ 10)
586 ⫾ 21cd
(n ⫽ 7)
654 ⫾ 28
(n ⫽ 9)
708 ⫾ 24
(n ⫽ 6)
661 ⫾ 30
(n ⫽ 8)
666 ⫾ 26
(n ⫽ 10)
678 ⫾ 32
(n ⫽ 9)
618 ⫾ 24c
(n ⫽ 7)
689 ⫾ 28
(n ⫽ 8)
758 ⫾ 29
(n ⫽ 6)
a
Values are expressed as mean ⫾ SE. PHN, passive Heymann nephritis; ACEi, angiotensin-converting enzyme inhibitor; AIIRA,
angiotensin II receptor antagonist.
b
P ⬍ 0.05 versus control.
c
P ⬍ 0.01 versus control.
d
P ⬍ 0.05 versus vehicle.
e
P ⬍ 0.01 versus vehicle.
f
P ⬍ 0.05 versus lisinopril.
Table 2. Time course of systolic blood pressure (mmHg) in PHN rats
Groups
PHN ⫹ vehicle
PHN ⫹ ACEi
PHN ⫹ ACEi ⫹ AIIRA
PHN ⫹ ACEi ⫹ AIIRA ⫹ cerivastatin
PHN ⫹ cerivastatin
Control
Values are expressed as mean ⫾ SE.
P ⬍ 0.05 versus control.
c
P ⬍ 0.01 versus control.
d
P ⬍ 0.05 versus vehicle and cerivastatin.
e
P ⬍ 0.01 versus vehicle and cerivastatin.
f
P ⬍ 0.01 versus lisinopril.
g
P ⬍ 0.05 versus vehicle.
a
b
0 mo
2 mo
(before
treatment)
4 mo
6 mo
8 mo
10 mo
122 ⫾ 3
(n ⫽ 10)
121 ⫾ 2
(n ⫽ 10)
118 ⫾ 2
(n ⫽ 10)
122 ⫾ 2
(n ⫽ 10)
122 ⫾ 3
(n ⫽ 10)
116 ⫾ 4
(n ⫽ 6)
127 ⫾ 2
(n ⫽ 10)
130 ⫾ 3
(n ⫽ 10)
129 ⫾ 3
(n ⫽ 10)
130 ⫾ 2
(n ⫽ 10)
129 ⫾ 2
(n ⫽ 10)
121 ⫾ 2
(n ⫽ 6)
133 ⫾ 3c
(n ⫽ 10)
93 ⫾ 2ce
(n ⫽ 10)
91 ⫾ 3ce
(n ⫽ 10)
83 ⫾ 1cef
(n ⫽ 8)
123 ⫾ 2g
(n ⫽ 10)
122 ⫾ 1
(n ⫽ 6)
144 ⫾ 3c
(n ⫽ 10)
94 ⫾ 3ce
(n ⫽ 10)
87 ⫾ 3ce
(n ⫽ 10)
85 ⫾ 2ce
(n ⫽ 7)
137 ⫾ 4b
(n ⫽ 9)
122 ⫾ 4
(n ⫽ 6)
148 ⫾ 4c
(n ⫽ 9)
85 ⫾ 2ce
(n ⫽ 10)
86 ⫾ 3ce
(n ⫽ 10)
85 ⫾ 2ce
(n ⫽ 7)
135 ⫾ 2cg
(n ⫽ 9)
121 ⫾ 2
(n ⫽ 6)
152 ⫾ 5c
(n ⫽ 8)
90 ⫾ 4ce
(n ⫽ 10)
90 ⫾ 3ce
(n ⫽ 9)
85 ⫾ 2ce
(n ⫽ 7)
137 ⫾ 3cg
(n ⫽ 8)
120 ⫾ 2
(n ⫽ 6)
J Am Soc Nephrol 13: 2898–2908, 2002
Angiotensin II Blockade Plus Statin Prevent Progressive Nephropathy
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Table 3. Time course of urinary protein excretion (mg/d) in PHN ratsa
Groups
PHN ⫹ vehicle
PHN ⫹ ACEi
PHN ⫹ ACEi ⫹ AIIRA
PHN ⫹ ACEi ⫹ AIIRA ⫹ cerivastatin
PHN ⫹ cerivastatin
Control
0 mo
2 mo
(before
treatment)
4 mo
6 mo
8 mo
10 mo
20 ⫾ 1.9
(n ⫽ 10)
21 ⫾ 1.2
(n ⫽ 10)
20 ⫾ 1.5
(n ⫽ 10)
23 ⫾ 0.7
(n ⫽ 10)
17 ⫾ 1.3
(n ⫽ 10)
21 ⫾ 2.2
(n ⫽ 6)
425 ⫾ 69c
(n ⫽ 10)
408 ⫾ 64c
(n ⫽ 10)
406 ⫾ 51c
(n ⫽ 10)
439 ⫾ 54c
(n ⫽ 10)
406 ⫾ 71c
(n ⫽ 10)
28 ⫾ 7
(n ⫽ 6)
625 ⫾ 94c
(n ⫽ 10)
274 ⫾ 86cd
(n ⫽ 10)
127 ⫾ 28ce
(n ⫽ 10)
55 ⫾ 14efg
(n ⫽ 8)
474 ⫾ 58ce
(n ⫽ 10)
32 ⫾ 6
(n ⫽ 6)
653 ⫾ 80c
(n ⫽ 10)
352 ⫾ 123c
(n ⫽ 10)
129 ⫾ 44ce
(n ⫽ 10)
59 ⫾ 22efg
(n ⫽ 7)
632 ⫾ 75c
(n ⫽ 10)
40 ⫾ 9
(n ⫽ 6)
700 ⫾ 70c
(n ⫽ 9)
344 ⫾ 117
(n ⫽ 10)
187 ⫾ 75e
(n ⫽ 10)
76 ⫾ 29ef
(n ⫽ 7)
711 ⫾ 87c
(n ⫽ 9)
49 ⫾ 9
(n ⫽ 6)
697 ⫾ 47c
(n ⫽ 8)
381 ⫾ 131
(n ⫽ 10)
272 ⫾ 86be
(n ⫽ 9)
92 ⫾ 20efh
(n ⫽ 7)
752 ⫾ 78c
(n ⫽ 8)
88 ⫾ 26
(n ⫽ 6)
Values are expressed as mean ⫾ SE.
P ⬍ 0.05 versus control.
c
P ⬍ 0.01 versus control.
d
P ⬍ 0.05 versus vehicle.
e
P ⬍ 0.01 versus vehicle.
f
P ⬍ 0.01 versus cerivastatin.
g
P ⬍ 0.05 versus lisinopril, lisinopril ⫹ L158,809.
h
P ⬍ 0.05 versus lisinopril⫹L158,809.
a
b
Table 4. Time course of serum creatinine (mg/dl) in PHN ratsa
Groups
PHN ⫹ vehicle
PHN ⫹ ACEi
PHN ⫹ ACEi ⫹ AIIRA
PHN ⫹ ACEi ⫹ AIIRA ⫹ cerivastatin
PHN ⫹ cerivastatin
Control
0 mo
2 mo
(before
treatment)
4 mo
8 mo
10 mo
0.60 ⫾ 0.03
(n ⫽ 10)
0.59 ⫾ 0.02
(n ⫽ 10)
0.59 ⫾ 0.02
(n ⫽ 10)
0.57 ⫾ 0.02
(n ⫽ 10)
0.55 ⫾ 0.02
(n ⫽ 10)
0.57 ⫾ 0.02
(n ⫽ 6)
0.81 ⫾ 0.02c
(n ⫽ 10)
0.86 ⫾ 0.03c
(n ⫽ 10)
0.82 ⫾ 0.02c
(n ⫽ 10)
0.84 ⫾ 0.03c
(n ⫽ 10)
0.81 ⫾ 0.04c
(n ⫽ 10)
0.64 ⫾ 0.01
(n ⫽ 6)
1.10 ⫾ 0.03c
(n ⫽ 10)
0.86 ⫾ 0.02cd
(n ⫽ 10)
0.82 ⫾ 0.03cd
(n ⫽ 10)
0.79 ⫾ 0.03cef
(n ⫽ 8)
1.12 ⫾ 0.03c
(n ⫽ 10)
0.61 ⫾ 0.03
(n ⫽ 6)
1.22 ⫾ 0.10c
(n ⫽ 9)
0.95 ⫾ 0.05c
(n ⫽ 10)
0.87 ⫾ 0.06bdf
(n ⫽ 10)
0.79 ⫾ 0.03cef
(n ⫽ 7)
1.12 ⫾ 0.12c
(n ⫽ 9)
0.63 ⫾ 0.04
(n ⫽ 6)
2.03 ⫾ 0.67c
(n ⫽ 8)
1.06 ⫾ 0.06c
(n ⫽ 10)
0.95 ⫾ 0.07bd
(n ⫽ 9)
0.86 ⫾ 0.03bef
(n ⫽ 7)
1.27 ⫾ 0.08c
(n ⫽ 8)
0.74 ⫾ 0.03
(n ⫽ 6)
Values are expressed as mean ⫾ SE.
P ⬍ 0.05 versus control.
c
P ⬍ 0.01 versus control.
d
P ⬍ 0.05 versus vehicle and cerivastatin.
e
P ⬍ 0.01 versus vehicle and cerivastatin.
f
P ⬍ 0.05 versus lisinopril.
a
b
macrophages (Chemicon, Temecula, CA) and rat CD8⫹ cell surface
glycoprotein on T-suppressor cells (OX8; PharMingen, Los Angeles,
CA). ED-1 antigen was stained on paraffin sections using an alkaline
phosphatase-Fast Red technique. CD8 staining was analyzed by indirect immunofluorescence technique. Fragments of renal tissues were
frozen in liquid nitrogen and cut at 3 ␮m using a Mikrom 500 O
cryostat (Walldorf, Germany). The sections were blocked with 1%
PBS/BSA, incubated overnight at 4°C with the primary antibody
(W3/25, 40 ␮g/ml; OX6, 5 ␮g/ml), washed with PBS, and then
incubated with Cy3-conjugated donkey anti-mouse IgG antibodies (5
␮g/ml in PBS; Jackson ImmunoResearch Laboratories, West Grove,
PA) for 1 h at room temperature. For each marker, positive cells were
counted in at least ten randomly selected high-power microscopic
fields (⫻400) per each animal.
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J Am Soc Nephrol 13: 2898–2908, 2002
Table 5. Serum lipid profile and serum transaminase levels in PHN ratsa
Groups
PHN ⫹ vehicle
PHN ⫹ ACEi
PHN ⫹ ACEi ⫹ AIIRA
PHN ⫹ ACEi ⫹ AIIRA ⫹ cerivastatin
PHN ⫹ cerivastatin
Control
Serum
Cholesterol
(mg/dl)
Serum
Triglycerides
(mg/dl)
ALT
(IU/L)
AST
(IU/L)
154 ⫾ 15b
(n ⫽ 8)
94 ⫾ 16c
(n ⫽ 10)
91 ⫾ 16d
(n ⫽ 9)
65 ⫾ 5d
(n ⫽ 7)
161 ⫾ 18b
(n ⫽ 8)
67 ⫾ 4
(n ⫽ 6)
367 ⫾ 25b
(n ⫽ 8)
241 ⫾ 68c
(n ⫽ 10)
163 ⫾ 35d
(n ⫽ 9)
153 ⫾ 45d
(n ⫽ 7)
392 ⫾ 44b
(n ⫽ 8)
186 ⫾ 21
(n ⫽ 6)
35 ⫾ 7
(n ⫽ 8)
34 ⫾ 5
(n ⫽ 10)
46 ⫾ 4
(n ⫽ 9)
44 ⫾ 3
(n ⫽ 7)
37 ⫾ 4
(n ⫽ 8)
51 ⫾ 5
(n ⫽ 6)
76 ⫾ 12
(n ⫽ 8)
79 ⫾ 13
(n ⫽ 10)
77 ⫾ 14
(n ⫽ 9)
81 ⫾ 8
(n ⫽ 7)
54 ⫾ 3
(n ⫽ 8)
70 ⫾ 3
(n ⫽ 6)
Values are expressed as mean ⫾ SE.
P ⬍ 0.01 versus control.
c
P ⬍ 0.05 versus vehicle.
d
P ⬍ 0.01 versus vehicle.
a
b
was labeled with ␣-32P dCTP by random-primed method. Hybridization was performed overnight in 0.25 mol/L Na2HPO4, pH 7.2, 7%
SDS. Filters were washed twice for 30 min with 20 mmol/L
Na2HPO4, pH 7.2, 5% SDS and two times for 10 min with 20 mmol/L
Na2HPO4, pH 7.2, 1% SDS at 65°C. Membranes were subsequently
probed with a glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
cDNA, taken as internal standard of equal loading of the samples on
the membrane. TGF-␤ mRNA optical density was normalized to that
of the constituently released GAPDH gene expression.
Statistical Analyses
Data of all the animals until death were included in the statistical
analyses. Results were expressed as mean ⫾ SEM and analyzed using
the nonparametric Mann Whitney test or Kruskal-Wallis test for
multiple comparisons as appropriate. The statistical significance level
was defined as P ⬍ 0.05.
Results
Systemic Parameters
Figure 1. ACE activity in kidney homogenate from passive Heymann
nephritis (PHN) rats given vehicle (n ⫽ 8), cerivastatin (n ⫽ 8),
angiotensin-converting enzyme inhibitors (ACEi) (n ⫽ 10), ACEi
plus angiotensin II (AngII) receptor antagonist (AIIRA) plus cerivastatin (n ⫽ 7), and in control rats (n ⫽ 6). Data are mean ⫾ SE. °P ⬍
0.05, °°P ⬍ 0.01 versus control; *P ⬍ 0.05, **P ⬍ 0.01 versus
vehicle.
Northern Blot Analyses
Total RNA was isolated from whole kidney tissue by the guanidium isothiocyanate/cesium chloride procedure. Twenty micrograms of total RNA were then fractionated on 1.6% agarose gel and
blotted onto synthetic membranes (Zeta-probe; Biorad, Richmond,
CA). A 0.45 kb EcoRI/HindIII fragment of human TGF-␤1 cDNA
from plasmid pUC18 was used to detect 2.5 kb transcript. The probe
By the end of the study, two rats with PHN died in the
vehicle group (months 6.5 and 9), one in the group given
lisinopril plus L-158,809 (month 8.5), three in the group given
lisinopril plus L-158,809 plus cerivastatin (months 2.5, 3, and
5.5), two in the group on cerivastatin alone (months 7 and 9).
All rats on lisinopril alone and controls were alive at 10 mo.
Food intake was comparable in PHN and control groups for
the entire study period. As shown in Table 1, rats with PHN
gained weight along the study; however, body weight was
numerically lower than controls, with a statistical significance
being observed for rats given ACEi plus AIIRA plus statin.
Rats with PHN exhibited an increase in SBP with respect to
controls (Table 2). Treatment with the ACEi alone or in combination with AIIRA or with AIIRA plus cerivastatin maintained SBP at values lower than those of vehicle group, and
even of controls. In rats on cerivastatin alone, SBP was lower
J Am Soc Nephrol 13: 2898–2908, 2002
Angiotensin II Blockade Plus Statin Prevent Progressive Nephropathy
2903
than in vehicle rats, which is consistent with the antihypertensive effect previously described for statins and attributed to
drug interaction with endothelial function or AII receptors
(36).
Renal Parameters
In rats with PHN, mean values of proteinuria exceeded 400
mg/d in all groups before treatment (Table 3). Administration
of lisinopril maintained over time urinary protein excretion at
values comparable to those measured before treatment and
numerically, although not significantly, lower than vehicle.
When lisinopril was combined with the AIIRA L-158,809,
more marked antiproteinuric effect was evident, with proteinuria values being consistently reduced with respect to pretreatment and significantly different from vehicle at all time points
considered. Remarkably, proteinuria was further lowered to
control levels when animals were treated with the triple therapy
of ACEi plus AIIRA plus cerivastatin. In PHN rats given
cerivastatin, proteinuria values were lower than those measured in vehicle group at 4 mo, thereafter they became
comparable.
Renal function, as evaluated by serum creatinine levels, was
progressively impaired in PHN rats given vehicle (Table 4). In
rats treated with lisinopril, mean value of serum creatinine was
numerically lower than in rats given vehicle. A difference in
serum creatinine was achieved when lisinopril was combined
with L-158,809 and even to a more significant extent when all
three drugs were administered together. Treatment with cerivastatin did not improve renal function.
Serum Cholesterol and Triglycerides
In PHN rats given vehicle, serum cholesterol and triglyceride levels were increased with respect to controls (Table 5).
Lisinopril alone or combined with L-158,809 significantly
reduced hypercholesterolemia and hypertriglyceridemia. More
remarkably, cholesterol and triglycerides in the group given the
triple therapy accounted for values within the control range.
Administration of cerivastatin alone had no effect.
Serum Transaminase Levels
In PHN rats, serum transaminase levels were not modified
by treatments (Table 5).
Renal ACE Activity
ACE activity in renal homogenate from PHN rats was elevated as compared with controls (Figure 1). In response to
lisinopril treatment renal ACE activity was inhibited by more
than 60%. The addition of cerivastatin to ACEi plus AIIRA led
to a further striking reduction in ACE activity, which decreased
below control values. Treatment with cerivastatin alone resulted in a 30% inhibition of ACE activity.
Renal Histology
Rats with PHN exhibited glomerular and tubulointerstitial
changes 2 mo after disease induction (Figure 2). At 10 mo
in PHN rats given vehicle, on average 60% of glomeruli
were affected by sclerotic changes (Figures 2 and 3).
Figure 2. Renal morphologic parameters evaluated at month 2 in PHN
(n ⫽ 5, first column) and control rats (n ⫽ 4, second column) and at
month 10 in PHN rats given vehicle (n ⫽ 8), cerivastatin (n ⫽ 8),
ACEi (n ⫽ 10), ACEi plus AIIRA (n ⫽ 9), ACEi plus AIIRA plus
cerivastatin (n ⫽ 7), and in control rats (n ⫽ 6). Data are mean ⫾ SE.
§P ⬍ 0.01 versus control (month 2); *P ⬍ 0.05, **P ⬍ 0.01 versus
vehicle; °P ⬍ 0.05, FP ⬍ 0.01 versus cerivastatin; ⫹P ⬍ 0.01 versus
ACEi; #P ⬍ 0.05 versus ACEi, ACEi⫹AIIRA, ⌬P ⬍ 0.05 versus
PHN at month 2 .
Tubulointerstitial damage consisted of interstitial fibrosis
and inflammation associated with tubular atrophy and eosinophilic casts in the tubular lumen. Lisinopril alone or
combined with L-158,809 significantly protected PHN rats
from glomerulosclerosis, tubular damage, and interstitial
inflammation, with respect to vehicle-rats. In rats given the
triple therapy, complete renoprotection was achieved, so
that glomerular and tubular morphology was comparable to
age-matched normal controls. Actually, renal injury documented in PHN at 2 mo was reversed by the multidrug
therapy. Treatment with cerivastatin had only a mild protective effect on renal damage.
2904
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J Am Soc Nephrol 13: 2898–2908, 2002
Figure 3. Light micrographs of sections of kidney cortex showing effects of drug treatments on renal structural changes in PHN. (A)
PHN⫹vehicle, (B) PHN⫹cerivastatin, (C) PHN⫹lisinopril, (D) PHN⫹lisinopril⫹L-158,809, (E) PHN⫹lisinopril⫹L-158,809⫹cerivastatin,
(F) age-matched control rats. Magnification, ⫻100.
Renal Expression of TGF-␤ mRNA
Figure 4. ED-1–positive monocytes/macrophages and CD-8 –positive
T cells infiltrating the interstitium of rats with PHN (n ⫽ 8) and the
effect of cerivastatin (n ⫽ 8), ACEi (n ⫽ 10), ACEi⫹AIIRA (n ⫽ 9),
ACEi⫹AIIRA⫹cerivastatin (n ⫽ 7). Data are mean ⫾ SE. HPF, high
power field; *P ⬍ 0.05, **P ⬍ 0.01 versus vehicle; °P ⬍ 0.05, #P ⬍
0.01 versus ACEi, ACEi⫹AIIRA, and cerivastatin.
Inflammatory Cell Infiltrates in Renal Interstitium
A massive infiltration of ED-1–positive monocytes/macrophages (Figure 4) and CD-8 –positive T cells (Figures 4 and 5)
was present in the renal interstitium of PHN rats given vehicle,
as evaluated at month 10. Lisinopril alone or combined with
L-158,809 limited the number of infiltrating cells. The degree
of cell infiltrates was decreased further by the combined administration of ACE inhibitor plus AIIRA plus statin. A tendency toward less accumulation of ED-1–positive cells was
observed in the renal interstitium of rats treated with cerivastatin in respect to vehicle rats, although statistical significance
was not reached.
Upregulation of TGF-␤1 mRNA was observed in the kidney
of PHN rats given vehicle (Figure 6). Densitometric analyses
of the autoradiographic signals showed a 4.8-fold increase in
TGF-␤1 transcript levels with respect to age-matched controls.
TGF-␤1 gene overexpression was partially reduced after the
administration of lisinopril alone or combined with L-158,809
(34 to 38% inhibition in respect to vehicle), but a statistical
significance was not achieved. By contrast, the TGF-␤1 signal
appeared significantly reduced after lisinopril plus L-158,809
plus cerivastatin treatment. In the rats given cerivastatin TGF␤1, gene upregulation was inhibited by 25% with respect to
vehicle.
Discussion
Results from this study demonstrate that in a severe model of
proteinuric nephropathy, which partly resembles advanced
phases of human disease, regression of proteinuria and complete protection of the kidney can be achieved by combined
administration of ACEi, AIIRA, and statin.
The current therapy for chronic proteinuric nephropathies is
ACEi that limit proteinuria and reduce GFR decline and risk of
end-stage renal disease more effectively than other antihypertensive treatments (14,37,38). Full remission of proteinuria,
however, is seldom obtained, and ACEi may be not effective to
the same degree in all individuals, particularly when therapy is
started late. For nonresponders, treatment procedure to remission and/or regression must include a multimodal strategy
(14,39). Here, we documented that lisinopril given from 2 to 10
J Am Soc Nephrol 13: 2898–2908, 2002
Angiotensin II Blockade Plus Statin Prevent Progressive Nephropathy
2905
Figure 5. Representative photomicrographs of sections of kidney cortex stained for detection of CD-8 –positive T cells, obtained at 10 mo from
PHN rats given vehicle (A), cerivastatin (B), lisinopril (C), lisinopril⫹L-158,809 (D), or lisinopril⫹L-158,809⫹cerivastatin (E) and from
age-matched control rats (F). Magnification, ⫻200.
mo after disease induction to PHN rats with heavy proteinuria
kept urinary protein excretion at levels that were both comparable to pretreatment and numerically, albeit not significantly,
lower than those of rats given no drug. Renal function ameliorated after ACEi but not to a significant extent. By contrast,
the addition of AIIRA therapy to lisinopril resulted in greater
antiproteinuric effect, being urinary protein excretion consistently reduced with respect to pretreatment values and significantly lower than vehicle rats at any time points considered.
Thus, blocking the receptor binding in concomitance with the
formation of AII further increased the antiproteinuric effect of
the ACEi alone and further protected against renal function
deterioration. When cerivastatin was added on top of ACE
inhibition and AT1 blockade, proteinuria regressed toward
normal values and renal failure was prevented. Cerivastatin
alone had effects on proteinuria only in the early phase of
treatment and partially decreased serum creatinine levels.
The mechanism(s) by which ACEi plus AIIRA, and to a
greater extent the addition of statin, lowered proteinuria in
PHN animals below the pretreatment levels, can be related to
the combined drugs’ actions on the glomerular filtration barrier
function. As suggested by several studies, both ACEi and
AIIRA reduce membrane pore dimensions and improve glomerular size-selectivity in experimental and human proteinuric
nephropathies (40 – 43). There is also evidence that ACEi preserved heparan sulfate proteoglycans in the glomerular basement membrane (GBM) of rats with adriamycin nephropathy
(44). In the PHN model, we documented that the early treatment with lisinopril preserved the frequency of epithelial slits
and prevented the associated loss of hydraulic permeability of
the GBM (45). Moreover, in the same model, blocking AII
synthesis or activity preserved the expression of nephrin, the
slit diaphragm protein in the podocytes (46). On the other hand,
preliminary data in 5/6 nephrectomized rats fed a high-cholesterol diet indicate that statins have the capability to preserve
anionic sites in the GBM (Suzuki T, personal communication),
that may account for the maximal antiproteinuric effect
achieved by adding cerivastatin to ACEi and AIIRA.
Data of a similar BP control among PHN rats receiving
either lisinopril or the combined therapies would weaken the
role for the BP lowering action in the superior protective
effects of the multidrug therapy. In fact, recent data have
shown that in PHN the early treatment with the antihypertensive drug lacidipine, at variance with lisinopril, failed to limit
proteinuria and renal damage, despite similar degree of BP
reduction (47).
Cerivastatin alone did not modify hypercholesterolemia of
PHN, a finding also described in other rat models, including
puromycin aminonucleoside nephrosis (48), mesangial proliferative nephritis (49), and AngII–induced renal injury (31).
However, addition of cerivastatin to AngII blocking agents
lowered serum cholesterol to normal levels in parallel to and as
a likely consequence of the strong antiproteinuric effect of
triple therapy (10).
An interesting finding of the current study is that the addition of cerivastatin on the background of ACE inhibition resulted in a dramatic decrease of renal ACE activity. Actually,
ACE activity was increased in the kidney of PHN rats and
could be inhibited by more than 60% after lisinopril. Adding
cerivastatin to ACEi plus AIIRA led to a further striking
2906
Journal of the American Society of Nephrology
Figure 6. (Top) Renal expression of TGF-␤ mRNA assessed at month
10 in age-matched control rats (n ⫽ 6) and in PHN rats given vehicle
(n ⫽ 8), cerivastatin (n ⫽ 8), ACEi (n ⫽ 10), ACEi⫹AIIRA (n ⫽ 9),
ACEi⫹AIIRA⫹cerivastatin (n ⫽ 7). Northern blot experiments were
performed using total RNA from whole kidney tissue of either separate or pooled samples for each group. Results shown are representative of pooled samples for each group. (Bottom) Densitometric
analysis of the autoradiographic signals for TGF-␤. Results shown are
mean ⫾ SE of separate animals for each group. The optical density of
the autoradiographic signals was quantitated and calculated as the
ratio of TGF-␤ to GAPDH mRNA. Results expressed as fold increase
over control (represented as 1) in densitometric arbitrary units. °P ⬍
0.01 versus control; *P ⬍ 0.05 versus other PHN groups.
J Am Soc Nephrol 13: 2898–2908, 2002
reduced AT1 receptor density in isolated platelets (52). It is
tempting to speculate that cerivastatin combined with lisinopril
and L-158,809, along with the possible effect of improving the
permselectivity of the glomerular barrier (as we stated above)
may contribute to achieve full inhibition of RAS, thereby
preventing local AngII generation and its deleterious effects
(19,20). Increased intrarenal synthesis of AII has been measured in experimental renal disease (53,54) and might contribute together with excess protein traffic in promoting tubulointerstitial inflammation and fibrosis (1). In this respect, AngII
immunoreactive material was detected in tubular cells after
subtotal nephrectomy in rats at sites of upregulation of TGF-␤
and type IV collagen mRNA (54).
In vitro and in vivo studies have consistently documented
that statins modulate intracellular signaling pathways responsible for inflammation and fibrosis (29 –31). We have recently
reported that in severe PHN the beneficial effect of combined
therapy of lisinopril and simvastatin against injury could be
attributed, at least in part, to further inhibition of MCP-1–
dependent interstitial inflammation by simvastatin (10). Here,
we extended our observations to TGF-␤, the crucial mediator
of fibrosis for which expression was also found to be reduced
by lovastatin in glomeruli of diabetic rats (55). Upregulation of
TGF-␤ mRNA of PHN kidney was inhibited by 25% after
cerivastatin and almost normalized after combined administration of ACEi plus AIIRA plus statin therapy. The finding that
renal structural integrity was fully preserved by triple therapy
clearly indicates that simultaneous blocking of pathways of
injury, including proteinuria, AII, and TGF-␤, eventually translates into both full prevention of progressive parenchymal
injury and preservation of renal function. Our data cannot
clearly unravel a hierarchy of protective mechanisms or establish which combination(s) of factors was most affected by the
multidrug treatment. However, maximization of the antiproteinuric action, presumably by mechanisms at the glomerular
level, appears to play an important role to achieve protection,
possibly in combination with the inhibitory effect on secondary
pathways, leading to inflammatory and immune cell accumulation and fibrosis.
In conclusion, our data suggest a possible future strategy to
induce remission of proteinuria as well as to lessen renal injury
and protect from loss of function in those patients who do not
fully respond to ACEi therapy.
Acknowledgments
reduction in ACE activity below control values. Notably, treatment with cerivastatin alone resulted in a 30% inhibition of
ACE activity. These data are in agreement with the observation
of an inhibitory effect of statins on ACE activity in a different
experimental setting, cardiac hypertrophy induced by hemodynamic overload in rats (50). That statins can directly interfere
with RAS is also suggested by findings that atorvastatin downregulated AT1 receptor mRNA expression either in cultured
vascular smooth muscle cells exposed to AngII or in aortic
segments of spontaneously hypertensive rats (51). Moreover,
in hypercholesterolemic patients, statin treatment effectively
We thank Dr. Marcella Pagnoncelli for animal care assistance. We
are also indebted to Drs. Flavio Gaspari and Roberta Donadelli for
helpful collaboration. Cerivastatin was provided by Dr. Hilmar
Bischoff, Bayer AG, Wuppertal, Germany. Lisinopril was kindly
provided by AstraZeneca, Basiglio, Milan, Italy, and L-158,809 by
Merck & Co., Inc., Rahway, NJ. Part of this study has been presented
at the 34th Annual Meeting of the American Society of Nephrology,
San Francisco, CA, October 13–17, 2001.
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See related editorial, “The Next Treatments of Chronic Kidney Disease: If We Find Them, Can We Test Them,” on pages
3024 –3026.