Nephrol Dial Transplant (2015) 30: 762–770
doi: 10.1093/ndt/gfu388
Advance Access publication 19 December 2014
Original Articles
Angiotensin II-mediated GFR decline in subtotal nephrectomy
is due to acid retention associated with reduced GFR
Donald E. Wesson1,2, Chan-Hee Jo3 and Jan Simoni4
1
Department of Internal Medicine, Baylor Scott and White Health, Temple, TX, USA, 2Texas A&M Health Sciences Center College of Medicine,
Temple, TX, USA, 3Department of Biostatistics, Baylor Scott and White Health, Temple, TX, USA and 4Department of Surgery, Texas Tech
University Health Sciences Center, Lubbock, TX, USA
Correspondence and offprint requests to: Donald E. Wesson; E-mail: [email protected]
A B S T R AC T
INTRODUCTION
Background. Angiotensin II (AII) mediates glomerular filtration
rate (GFR) decline in animals with subtotal nephrectomy (Nx),
but the mechanisms for increased AII activity are unknown.
Because reduced GFR of Nx is associated with acid (H+) retention that increases kidney AII, AII-mediated GFR decline might
be induced by H+ retention.
Methods. We measured GFR and kidney microdialyzate H+
and AII content in Sham and 2/3 Nx rats in response to amelioration of H+ retention with dietary NaHCO3, to AII receptor
antagonism and to both.
Results. GFR was lower in Nx than that in Sham. Nx but not
Sham GFR was lower at Week 24 than that at Week 1. Despite
no differences in plasma acid–base parameters or urine net
acid excretion, kidney H+ content was higher in Nx than that
in Sham, consistent with H+ retention. Plasma and kidney microdialyzate AII were higher in Nx than that in Sham and
dietary NaHCO3 reduced each in Nx but not in Sham. AII receptor antagonism was associated with higher Week 24 GFR
in Nx with H+ retention but not in Sham or in Nx in which
H+ retention had been corrected with dietary NaHCO3. Week
24 GFR after dietary NaHCO3 was higher than after AII receptor antagonism. Week 24 GFR was not different after adding
AII receptor antagonism to dietary NaHCO3.
Conclusions. AII-mediated GFR decline in 2/3 Nx was induced
by H+ retention and its amelioration with dietary HCO3 conserved GFR better than AII receptor antagonism in this CKD
model. H+ retention might induce AII-mediated GFR decline in
patients with reduced GFR, even without metabolic acidosis.
Animals with the 5/6 nephrectomy (Nx) model of chronic kidney
disease (CKD) have progressive glomerular filtration rate (GFR)
decline [1, 2] mediated through angiotensin II (AII) receptors [3].
Because anti-AII drug therapy helps conserve GFR in patients
with reduced GFR [4, 5], AII appears to mediate GFR decline in
CKD patients. AII also mediates increased nephron acidification
in 5/6 Nx [6, 7] and 2/3 Nx [8]. Increased acidification in 2/3 Nx
allows higher per nephron H+ excretion, thereby avoiding metabolic acidosis in this setting of reduced overall GFR [8]. So,
further GFR decline in animals with reduced GFR might be a
pathophysiologic consequence of kidney physiologic mechanisms
to maintain steady-state H+ excretion in the face of unchanged
dietary H+. Specifically, kidney AII induced by H+ retention in 2/
3 Nx [8] yields the short-term physiologic benefit of increased per
nephron acidification but might also yield the longer-term pathophysiologic consequence of GFR decline through AII receptors. If
so, reducing H+ retention with dietary HCO3 might lower kidney
AII and thereby help conserve GFR. The present study tested the
hypothesis that the progressive GFR decline of 2/3 Nx is mediated
by H+ retention through AII receptors.
M AT E R I A L S A N D M E T H O D S
Animals, diet and protocol
Eight-week-old male and female Munich-Wistar rats (Harlan
Sprague-Dawley, Houston, TX) ate standard chow (Prolab RMH
2500 with 23% protein of various sources, Purina Labs, St Louis,
MO) before kidney mass reduction surgery (below). Because
daily NaHCO3 slowed GFR decline in patients with CKD stage-2
Keywords: aldosterone, angiotensin II, bicarbonate, chronic
kidney disease, endothelin, microdialysis
© The Author 2014. Published by Oxford University Press
on behalf of ERA-EDTA. All rights reserved.
762
Kidney mass reduction
Nx was induced by surgical removal of ∼2/3 of kidney mass
in two stages and chronic vascular lines inserted [11]. One
week after the second surgery during which animals ate the
described experimental diet, GFR was measured in conscious
Nx and Sham by slope of the decrease in plasma concentration
of intravenously infused 3H-inulin over 180 min [15].
Microdialysis to compare kidney cortical H+ and AII
Twenty-three weeks after the second surgery during which
animals ate the experimental diet, a microdialysis catheter was
inserted into the kidney cortex of Nx and Sham through a flank
incision [11, 12]. Relative H+ retention was determined by comparing H+ content ([H+] times dialyzate volume) between collected and infused dialyzate [11, 12]. Kidney cortical AII
content was similarly compared as done previously [8].
Microdialysis of kidney cortex was done in comfortably restrained, conscious animals 7 days after microdialysis catheter
insertion (24 weeks after kidney mass reduction) using a dialyzate that would gain H+ when dialyzed against kidney
cortex with higher-than-Sham H+ content and would lose H+
if kidney cortex H+ were less [11, 12]. Three 20-min collection
periods were done in eight animals in each group. Anaerobically obtained, collected and infused dialyzates were analyzed
for pH (Micro flow through pH monitor, Lazar Research Labs,
Los Angeles, CA), PCO2 (Micro flow through CO2 probe,
Lazar Research Labs) and total CO2 (TCO2) by flow-through
ultrafluorometry [10].
Whole blood and plasma parameters
Immediately after microdialysis (24 weeks after kidney mass
reduction), 0.35 mL of carotid arterial blood was slowly
removed for arterial blood gases, plasma total CO2 (by flowthrough ultrafluorometry), electrolytes and AII from awake,
calm and gently restrained animals. It was replaced with an
equivalent blood volume from a paired, identically treated
animal. The animal was then returned to its metabolic cage for
an additional 1 week. GFR was repeated, now 24 weeks after
initial GFR measurement and 25 weeks after kidney mass reduction. Additional studies were done in two groups of separate
animals to determine the effect of valsartan discontinuation on
GFR. In Group 1, valsartan was continued for the entire 24
weeks as described. In Group 2, valsartan was stopped at Week
23. In both groups, GFR was measured at Weeks 1, 23 and 24.
Urine NAE
Six days after insertion of the microdialysis catheter, urine
NAE was measured [16] in a 24-h sample in eight animals
each of control and experimental groups kept in metabolic
cages.
Analytical methods
Dialyzate and plasma AII were measured as described previously [8] with an RIA kit (SPI-BIO, France) after storage at
−80°C in tubes containing EDTA, Pepstatin, enalaprilate and
1,10-phenanthroline and evaporated to dryness, as described
previously [17].
Calculations
Urine NAE was the mean for each animal group. Net dialyzate H+ addition was averaged for the three collection
periods per animal that was then averaged for each animal in
each group [11, 12]. Positive values for net H+ addition
Acid retention and angiotensin II-mediated GFR decline
763
ORIGINAL ARTICLE
eGFR without metabolic acidosis [9], we developed an animal
model of such subjects. Unlike conscious 5/6 Nx that had lower
arterial plasma total CO2 (PTCO2) than Sham [2] by ultrafluorometry [10], PTCO2 by ultrafluorometry was comparable in conscious 2/3 Nx and Sham despite greater kidney and skeletal
muscle H+ content in 2/3 Nx [8, 11, 12]. So, we used 2/3 Nx to
determine whether H+ retention mediated GFR decline and did
so through AII.
After kidney mass reduction, animals ate minimum electrolyte diets with 20% protein as casein (ICN Nutritional Biochemicals, Cleveland, OH) and drank distilled H2O. We compared
GFR at 1 and 24 weeks after nephron mass reduction rather
than 12 weeks [2] to allow for greater GFR decline. To examine
AII as a potential mediator of GFR decline in 2/3 Nx with
reduced GFR and H+ retention but without metabolic acidosis,
other animals received oral NaHCO3 to reduce H+ retention as
discussed. We assessed H+ retention with in vivo microdialysis
of kidney cortex [11–13]. Like Sham, Nx eating diets with 150
μM/g diet/day of added NaHCO3 had no net H+ addition to microdialyzate [11] (see below). Previous studies [2] showed that
H+ retention in animals ingesting 150 μM/g diet/day of added
NaCl was not different from baseline Nx. Because standard
chow in our laboratory contained 150 μM NaCl/g diet, the
dietary Na+ load was comparable with that routinely received in
our laboratory. Nx and similar weight controls ate 17.5 ± 0.9
versus 18.4 ± 0.9 g/day, respectively, (n = 4, P = 0.09) and so all
received 17 g/day.
Because valsartan has greater affinity for the AT 1 receptor
than losartan [13], some animals received valsartan (Novartis
Pharmaceuticals, East Hanover, NJ), mixed with diet at 10 mg/kg
body weight (bw)/day, a dose that reduced AII-induced aldosterone synthase mRNA in rats [14]. As detailed previously [8], we
attempted to determine the maximum tolerated valsartan dose.
Although animals tolerated valsartan as high as 25 mg/kg given
alone, tail-cuff blood pressure was considerably lower (81.0 ± 5.0
versus 99.2 ± 5.9 n = 4, P = 0.11) when this valsartan dose was
combined with darusentan and eplerenone as dictated by one
protocol arm (see below). Importantly, GFR at Week 24 was not
different between 2/3 Nx given 25 mg/kg bw/day compared with
10 mg/kg bw/day valsartan alone (2179 ± 209 versus 2150 ± 202
µL/min, respectively, n = 4 animals each, P = 0.86), supporting
that 10 mg/kg bw/day yielded maximum GFR preservation.
Because earlier studies showed that GFR decline in 2/3 Nx was
mediated in part through endothelin A and aldosterone receptors
[12], some animals received valsartan along with the endothelin
A receptor antagonist darusentan (Knoll, AG, Ludwigshafen/
Rhein, Germany) mixed with diet at 20 mg/kg bww/day and
the aldosterone antagonist eplerenone (Pfizer, New York, NY)
in diet at 100 mg/kg bw/day to determine whether these
two receptor antagonist provided additional GFR conservation to
valsartan.
indicated greater H+ content in collected dialyzate compared
with infused dialyzate (i.e. H+ gain) and negative ones indicated lower H+ content (i.e. H+ loss).
Statistical analysis
Continuous variables were first examined for normality and
non-parametric tests such as a Kruskal–Willis test for more
than two groups and a Wilcoxon ranks-sum test for paired
samples were considered. The changes of GFR from pre to
post for each group were described by mean and SD and they
were considered with a one-sample t-test. The differences
among treatments were considered with one-way ANOVA followed by post hoc Tukey’s test. Data management and statistical analysis were performed using SAS software (version 9.3,
SAS Institute, Cary, NC) and graphs were created using R 3.0
(R Core Development Team, 2013). A P-value of <0.05 indicates a statistical significance.
ORIGINAL ARTICLE
R E S U LT S
Plasma acid–base parameters and K+ were not different
between Nx and Sham but Nx had higher tail-cuff blood pressure as in Table 1. Microdialyzate H+ addition was higher in
Nx than that in Sham, consistent with H+ retention in Nx.
Also, Nx had higher plasma AII and higher microdialyzate
AII. Nx and Sham urine net acid excretion (UNAE) was not different but Nx had lower urine excretion of NH+4 and HCO3.
Table 2 shows the effects of oral valsartan (Val), oral
NaHCO3 (HCO3) and their combination (Val + HCO3), on
the indicated parameters within treatment groups (*) and for
treatments within Nx and Sham across the three treatment
groups (**). Table 3 shows the intra-group comparisons for
the data given in Table 2. With the exception of higher plasma
and microdialyzate AII in treated animals, valsartan and
NaHCO3 did not affect parameters, including microdialyzate
H+, in Sham. In contrast, plasma pH, PCO2, total CO2 and
Table 1. Laboratory values for Sham versus Nx (n = 8 animals each for
Sham and Nx)
Arterial TCO2 (mM)
Arterial pH
Arterial PCO2 (mmHg)
Arterial K+ (meq/L)
Plasma AII (fmoles/mL)
Microdialyzate H+
addition (fmoles/mL)
Microdialyzate AII
addition (fmoles/mL)
Urine NAE (mM/day)
Urine NH+4 (mM/day)
Urine TA (meq/day)
Urine HCO3 (mM/day)
Tail-cuff BP (mmHg)
Sham
Nx
P-value
25.2 ± 0.7
7.40 ± 0.01
40.2 ± 1.0
4.0 ± 0.1
91.5 ± 15.0
124.5 ± 55.5
24.8 ± 0.6
7.40 ± 0.01
39.8 ± 0.9
4.0 ± 0.2
114.6 ± 14.1
466.1 ± 116.7
0.33
0.43
0.34
0.74
0.01
<0.01
93.1 ± 33.0
144.3 ± 25.7
<0.01
3.1 ± 0.3
1.7 ± 0.2
1.5 ± 0.2
0.09 ± 0.02
98.4 ± 3.8
2.9 ± 0.3
1.5 ± 0.1
1.4 ± 0.3
0.02 ± 0.01
110.4 ± 4.0
0.16
0.03
0.37
<0.01
<0.01
Values are means ± SD.
Sham, sham operated animals, Nx, 2/3 nephrectomy; TCO2, total CO2; NAE, net acid
excretion; urine NH+4 , urine ammonium excretion; urine TA, urine titratable acid
excretion; Urine HCO3, urine HCO3 excretion; AII, angiotensin II; microdialyzate,
effluent microdialyzate from microdialysis of the kidney cortex.
764
UNAE were lower in Nx given valsartan compared with Nx not
given valsartan. Lower UNAE in valsartan-treated compared
with valsartan-untreated Nx was due mostly to lower NH4 þ
excretion (P < 0.03). Like Sham, valsartan-treated Nx had
higher plasma and higher microdialyzate AII. Microdialyzate
H+ was not different in Nx given valsartan compared with
those not given valsartan.
Table 2 also shows that Nx given NaHCO3 compared with
Nx not given NaHCO3 had lower microdialyzate H+, consistent with lower H+ retention. Nx given NaHCO3 also had lower
plasma AII, lower microdialyzate AII and lower UNAE than Nx
without NaHCO3, but remaining parameters were not different. Nx given valsartan and NaHCO3 compared with
NaHCO3 alone had higher plasma and microdialyzate AII, but
the remaining parameters, including microdialyzate H+, were
not different. Tail-cuff blood pressure was lower in Nx given
valsartan than that in baseline (P < 0.01) and blood pressure
was higher in Nx given NaHCO3 than that in baseline and valsartan-treated Nx (P < 0.01). Blood pressure in Nx given valsartan and NaHCO3 was lower than those given NaHCO3
alone (P < 0.03).
Figure 1 shows that GFR was lower in Nx than in Sham at
both time points (Weeks 1 and 24) as expected. Although
Sham GFR was not different between Week 24 and Week 1
(4083 ± 163 versus 4139 ± 269 µL/min, respectively, P = 0.42),
GFR in Nx was lower at Week 24 than that at Week 1
(1700 ± 159 versus 2562 ± 143 µL/min, respectively, P < 0.01).
Figure 2 shows that neither valsartan nor NaHCO3 affected
Week 24 GFR in Sham. In contrast, Figure 3 shows that Nx
had lower GFR at Week 24 compared with Week 1 for baseline
(1644 ± 135 versus. 2526 ± 80 µL/min, respectively, P < 0.01),
after valsartan (2128 ± 84 versus 2515 ± 119 µL/min, respectively, P < 0.01) and after NaHCO3 (2349 ± 152 versus
2542 ± 143 µL/min, respectively, P < 0.01). Consequently, valsartan and NaHCO3 helped conserve GFR in Nx with H+ retention but had no GFR effect in Sham without H+ retention.
In Figure 3, GFR at Week 24 of Nx given valsartan was higher
than Nx GFR at Week 24 of baseline Nx (P < 0.01) and GFR at
Week 24 of Nx given NaHCO3 was higher than Nx GFR at
Week 24 of Nx given valsartan (P < 0.01).
Studies in Figure 4 determined whether valsartan additionally preserved GFR in Nx given NaHCO3. GFR was lower at
Week 24 than Week 1 in Nx given valsartan (2153 ± 115
versus 2526 ± 80 µL/min, respectively, P < 0.01), in Nx given
NaHCO3 (2333 ± 119 versus 2537 ± 94 µL/min, respectively,
P < 0.01) and in Nx given valsartan + NaHCO3 (2308 ± 80
versus 2530 ± 82 µL/min, respectively, P < 0.01). GFR at Week
24 of Nx given NaHCO3 was higher than GFR of Nx given valsartan at Week 24 (P < 0.01) but GFR at Week 24 of Nx given
valsartan + NaHCO3 was no different than GFR of Nx given
NaHCO3 alone (P = 0.89).
Because GFR decline in 2/3 Nx was mediated through endothelin A and aldosterone receptors [12], studies in Figure 5
explored whether adding the endothelin A receptor antagonist
darusentan and the aldosterone antagonist eplerenone provided GFR preservation additional to valsartan. Figure 5
shows lower GFR at Week 24 than that at Week 1 in Nx given
NaHCO3 (2352 ± 67 versus 2572 ± 60 µL/min, respectively,
D. E. Wesson et al.
Table 2. Comparisons between Nx and Sham and across groups (n = 8 animals each for Sham and Nx)
Baseline
P-value*
P-value*
HCO3
P-value*
Valsartan + HCO3
P-value**
23.7 ± 0.4
25.0 ± 0.2
<0.01
25.1 ± 0.4
25.0 ± 0.3
0.66
25.0 ± 0.3
<0.01
0.35
7.388 ± 0.005
7.401 ± 0.005
<0.01
7.404 ± 0.006
7.403 ± 0.005
0.79
7.403 ± 0.005
<0.01
0.78
37.7 ± 0.5
39.8 ± 0.6
<0.01
40.3 ± 0.9
39.4 ± 0.7
0.04
40.1 ± 0.7
<0.01
0.42
4.1 ± 0.1
4.0 ± 0.1
0.25
4.0 ± 0.1
4.0 ± 0.1
0.79
4.0 ± 0.1
0.18
0.51
163.3 ± 23.2
126.4 ± 15.8
<0.01
93.0 ± 22.5
83.1 ± 12.7
0.3
157.0 ± 18.3
<0.01
<0.01
757.4 ± 344.1
26.8 ± 27.7
<0.01
23.3 ± 74.2
21.1 ± 38.7
0.94
15.3 ± 50.6
<0.01
0.83
361.6 ± 94.8
148.3 ± 16.6
<0.01
100.1 ± 23.2
80.5 ± 9.0
0.04
359.1 ± 109.4
<0.01
<0.01
2.61 ± 0.23
3.01 ± 0.17
<0.01
1.09 ± 0.13
1.07 ± 0.12
1.28 ± 0.10
1.63 ± 0.12
<0.01
0.49 ± 0.09
0.47 ± 0.05
0.51
<0.01
<0.01
1.35 ± 0.15
1.51 ± 0.09
0.02
0.80 ± 0.03
0.83 ± 0.12
0.46
<0.01
<0.01
0.02 ± 0.01
0.13 ± 0.04
<0.01
0.20 ± 0.05
0.23 ± 0.07
0.36
<0.01
<0.01
101.6 ± 2.5
94.4 ± 3.3
<0.01
123.6 ± 4.7
98.1 ± 3.8
<0.01
<0.01
<0.01
108.1 ± 3.4
ORIGINAL ARTICLE
Arterial TCO2 (mM)
Nx
24.9 ± 0.3
0.59
Sham
24.8 ± 0.6
Arterial pH
Nx
7.403 ± 0.006
0.97
Sham
7.403 ± 0.007
Arterial PCO2 (mmHg)
Nx
39.8 ± 0.7
0.65
Sham
39.7 ± 0.6
Arterial K+ (meq/L)
Nx
4.0 ± 0.1
0.36
Sham
3.9 ± 0.1
Plasma AII (fmoles/mL)
Nx
121.1 ± 19.5
<0.01
Sham
90.0 ± 21.2
Microdialyzate H+ addition (fmoles/mL)
Nx
622.4 ± 128.8
<0.01
Sham
29.9 ± 16.8
Microdialyzate AII addition (fmoles/mL)
Nx
146.6 ± 26.0
<0.01
Sham
88.3 ± 14.8
Urine NAE (mM/day)
Nx
2.85 ± 0.31
0.16
Sham
3.09 ± 0.32
Urine NH4 þ (mM/day)
Nx
1.46 ± 0.12
<0.01
Sham
1.70 ± 0.12
Urine TA (meq/day)
Nx
1.42 ± 0.11
0.03
Sham
1.54 ± 0.10
Urine HCO3 (mM/day)
Nx
0.02 ± 0.02
<0.01
Sham
0.12 ± 0.04
Tail-cuff BP (mmHg)
Nx
109.9 ± 5.6
<0.01
Sham
98.3 ± 2.5
Valsartan
<0.01
0.08
*P-value is computed for Nx versus Sham within treatment.
**P-value is for treatments within Nx and Sham separately.
P < 0.01), in Nx given valsartan (2155 ± 64 versus 2563 ± 63
µL/min, respectively, P < 0.01) and in Nx given the three drugs
(2249 ± 60 versus 2550 ± 56 µL/min, respectively, P < 0.01).
GFR at Week 24 of Nx given valsartan and Nx given the three
drugs were each lower than Nx given NaHCO3 (P < 0.02). In
addition, GFR at Week 24 for Nx given the three drugs was
higher than Nx given valsartan (P < 0.02).
Because in previous studies 2/3 Nx given HCO3 had better
GFR conservation than those given darusentan and eplerenone combined and that these antagonists added no GFR
preservation to oral HCO3 [12], studies in Figure 6 determined
whether oral NaHCO3 provided GFR conservation in addition
to that provided by the three antagonists given together.
Figure 6 shows lower GFR at Week 24 than that at Week 1 in
Nx given NaHCO3 (2415 ± 54 versus 2576 ± 53 µL/min, respectively, P < 0.01), in Nx given the three drugs (2287 ± 29
versus 2537 ± 67 µL/min, respectively, P < 0.01) and in Nx
given the three drugs + NaHCO3 (2410 ± 62 versus 2541 ± 47
µL/min, respectively, P < 0.01). GFR at Week 24 of Nx given
the three drugs was lower than Nx given NaHCO3 (P < 0.01)
and lower than Nx given the three drugs + NaHCO3 (P < 0.01).
GFR was not different between Nx given NaHCO3 and those
given the three drugs + NaHCO3 (P = 0.98).
Because anti-AII drugs might lower GFR in some patients
with reduced GFR [5], we explored whether GFR increased in
animals chronically taking valsartan after the drug was
stopped. Table 4 shows GFR measurements of two groups of
separate animals at Weeks 1, 23 and 24. Group 1 continued
valsartan throughout the 24-week protocol. In contrast, Group
2 had valsartan stopped at Week 23 and GFR was measured 1
week later. GFR at Week 23 was lower than that at Week 1 for
both groups. Week 24 GFR was not different from Week 23
GFR in Group 1 but Week 24 was higher than Week 23 GFR
in Group 2 (P < 0.05, paired t). Nevertheless, there was no difference in Week 24 GFRs between Groups 1 and 2.
DISCUSSION
The present study showed that AII receptor antagonism preserved GFR in Nx with H+ retention but did not do so in
Sham that had no H+ retention nor in Nx in which H+
Acid retention and angiotensin II-mediated GFR decline
765
Table 3. Multiple intra-group comparisons
Multiple comparisons
Baseline versus
Valsartan
HCO3
Valsartan + HCO3
HCO3
Valsartan + HCO3
Valsartan + HCO3
0.746
0.888
<0.001
<0.001
0.991
<0.001
0.948
1.000
<0.001
<0.001
0.959
<0.001
0.491
0.836
<0.001
<0.001
0.933
0.002
0.001
0.493
0.056
0.700
<0.001
0.010
<0.001
<0.001
<0.001
0.932
<0.001
<0.001
1.000
<0.001
<0.001
0.602
0.514
1.000
<0.001
0.394
<0.001
<0.001
0.359
<0.001
<0.001
0.835
<0.001
<0.001
0.989
0.002
0.002
<0.001
<0.001
F I G U R E 1 : Box plots comparing whole-kidney GFR (µL/min) of
animals with 2/3 surgical nephron mass reduction (Nx, n = 8) with
sham-operated animals (Sham, n = 8), Week 1 and Week 24 after
surgery. The dark horizontal line represents the median and the box
encompasses values within the 25th percentile above and the 25th
below the median. *P < 0.05 versus respective Week 1 value; +P < 0.05
versus respective Sham.
retention had been corrected with NaHCO3. Because AII receptor antagonism had no effect on H+ retention, its GFR
preservation appeared not to be mediated through reduced H+
retention. In addition, Nx had higher than Sham microdialyzate AII that was reduced when H+ retention was corrected
with NaHCO3, supporting that amelioration of H+ retention
with NaHCO3 (but not with NaCl as in previous studies [12])
lowered kidney AII. Furthermore, NaHCO3 did not affect
follow-up GFR in Sham. These data affirm earlier studies
766
HCO3 versus
<0.001
ORIGINAL ARTICLE
Nx
Sham
Nx
Sham
Nx
Sham
Nx
Sham
Nx
Sham
Nx
Sham
Nx
Sham
Nx
Sham
Nx
Sham
Nx
Sham
Nx
Sham
Valsartan versus
<0.001
<0.001
showing that H+ retention mediates GFR decline in the 2/3 Nx
model of CKD [12] and show, for the first time, that this detrimental effect of H+ retention on GFR is mediated through AII
receptors.
Adding endothelin A and aldosterone receptor antagonism
to AII receptor antagonism preserved GFR better than AII
receptor antagonism alone, supporting additive roles for endothelin and aldosterone in the GFR decline of 2/3 Nx [12].
Because AII stimulates kidney production of endothelin [18]
and aldosterone [19], AII receptor antagonism alone might
inhibit kidney endothelin and/or aldosterone secretion and
thereby inhibit contributions of these two agents to GFR
decline. Nevertheless, increased extracellular H+ stimulates secretion of endothelin from kidney microvascular endothelial
cells [20] and of aldosterone from adrenal cells [21]. Consequently, H+ retention in 2/3 Nx might increase kidney levels of
endothelin and aldosterone independent of AII and therefore
might explain why endothelin A and aldosterone receptor antagonism provided added GFR conservation to AII receptor
antagonism.
The present study shows a small but significant GFR increase 1 week after valsartan that had been administered for 23
of the 24-week protocol was discontinued. These data are consistent with the small hemodynamic effect of anti-AII drugs to
decrease GFR in patients [5]. Nevertheless, the 24-week GFR
was not different between animals in which valsartan had
been stopped for 1 week compared with animals in which valsartan was continued for 24 weeks. These data support that
any anti-AII effect of valsartan to decrease GFR was small and
that the 24-week GFR of animals that continued valsartan for
D. E. Wesson et al.
F I G U R E 2 : Box plots comparing whole-kidney GFR (µL/min) of Sham (n = 8) animals at Week 1 and at Week 24 after 23 weeks of no add-
itional intervention (baseline), oral valsartan, oral NaHCO3 or oral valsartan + oral NaHCO3.
F I G U R E 5 : Box plots comparing whole-kidney GFR of 2/3 Nx at
(n = 8) at Week 1 and at Week 24 after 23 weeks of baseline, oral valsartan or oral NaHCO3. The circle below the box plot in the Week 24
Nx + HCO3 group represents a value that is greater than the 25th percentile below the median. *P < 0.05 versus respective Week 1 value;
+
P < 0.05 versus respective Nx + baseline; &versus respective Nx + Val.
Week 1 and at Week 24 after 23 weeks of oral NaHCO3 (n = 8), oral
valsartan (n = 8) or oral valsartan + darusentan + eplerenone (three
drugs) (n = 8). *P < 0.05 versus respective Week 1 value; +P < 0.05
versus respective Nx + HCO3; &versus respective Nx + Val.
F I G U R E 4 : Box plots comparing whole-kidney GFR of 2/3 Nx at
Week 1 and at Week 24 after 23 weeks of oral valsartan (n = 8), oral
NaHCO3 (n = 8) or oral valsartan + oral NaHCO3 (n = 8). The circle
below the box plot in the Week 24 Nx + Val group represents a value
that is greater than the 25th percentile below the median. *P < 0.05
versus respective Week 1 value; +P < 0.05 versus respective Nx + Val;
&
versus respective Nx + HCO3.
24 weeks is reflective of the degree of GFR conservation
induced by valsartan and that there is no greater GFR conservation that is being concealed by such a hemodynamic effect.
F I G U R E 6 : Box plots comparing whole-kidney GFR of 2/3 Nx at
Week 1 and at Week 24 after 23 weeks of oral NaHCO3 (n = 8), oral
three drugs (n = 8) or oral three drugs + HCO3 (n = 8). The circle
above the box plot in the Week 1 Nx + 3 drugs group represents a
value that is greater than the 25th percentile above the median.
*P < 0.05 versus respective Week 1 value; +P < 0.05 versus respective
Nx + HCO3; &versus respective three drugs.
Pharmacologic kidney protection for patients with reduced
GFR is currently done with antagonists of AII and aldosterone,
and endothelin antagonists are being explored as kidney
Acid retention and angiotensin II-mediated GFR decline
767
ORIGINAL ARTICLE
F I G U R E 3 : Box plots comparing whole-kidney GFR of 2/3 Nx
Table 4. Effect of valsartan discontinuation on GFR
Group 1 (µL/min)
Group 2 (µL/min)
Week 1
Week 23
Week 24
(+) valsartan
2543 ± 134
(+) valsartan
2554 ± 103
(+) valsartan
2135 ± 121*
(+) valsartan
2140 ± 128*
(+) valsartan
2142 ± 68*
(−) valsartan
2170 ± 40*+
ORIGINAL ARTICLE
*P < 0.05, computed versus Week 1, ANOVA.
+
P < 0.05, computed versus Week 23, paired t.
protection agents. The present study supports that H+ retention
associated with reduced GFR is a major contributor to the progressive GFR decline that follows the subtotal nephrectomy experimental model of CKD with reduced GFR seen in patients.
Previous studies from our laboratory show that H+ retentioninduced increased kidney levels of endothelin and aldosterone
contribute to GFR decline in the 2/3 Nx model of CKD with
reduced GFR [12]. The present study additionally shows a
quantitatively more important role for AII induced by H+ retention in subsequent GFR decline that occurs in this CKD model.
Furthermore, amelioration of H+ retention with oral NaHCO3
preserved GFR better than combined AII, endothelin A and aldosterone receptor antagonism. In addition, combined three-receptor antagonism provided no additional GFR preservation
when added to NaHCO3. Together, the present and previous
studies support that H+ retention mediates GFR decline in 2/3
Nx in part through AII, endothelin and aldosterone receptors
but that other mechanisms also contribute. Mechanisms
induced by H+ retention that might also mediate GFR decline
in Nx include oxidative stress [22] and complement activation
[1, 23]. The data collectively support further exploration of
dietary HCO3 as a kidney protection intervention that might be
better tolerated, less expensive and possibly more effective than
pharmacologic antagonists.
Contrary to our previous studies in which GFR of 2/3 Nx
given dietary alkali Ca(HCO3)2 was not different between
Week 24 and Week 1 [11], GFR of 2/3 Nx in the present study
given dietary alkali NaHCO3 to lower H+ retention to Sham
levels had GFR that was slightly but significantly lower at
Week 24 than that at Week 1. We used Ca(HCO3)2 as the
alkali salt in our previous studies of 5/6 Nx, because tail-cuff
blood pressure was much higher in 5/6 Nx given NaHCO3
than Ca(HCO3)2 (155 ± 8 versus 117 ± 6, n = 8, P < 0.01) [11].
We chose NaHCO3 as the alkali salt because it is the more
likely salt to be used for dietary acid reduction [24] and shows
GFR preservation in patients with reduced GFR but no metabolic acidosis [9]. In the present study, tail-cuff blood pressure
was higher in 2/3 Nx given NaHCO3 compared with that in
not given NaHCO3 (Tables 2 and 3) and was lower in 2/3 Nx
given valsartan with NaHCO3. Because blood pressure was
higher in 2/3 Nx of the present study given NaHCO3 than that
in 2/3 Nx previously given Ca(HCO3)2, we hypothesize that
higher blood pressure in the present study limited GFR conservation as has been described in CKD patients [25].
The mechanism of H+ retention in the setting of reduced
GFR is not clear. This phenomenon appears to be systemic in
nature, not just in the kidney cortex as reported presently, but
is also present in skeletal muscle of 2/3 Nx animals by
768
microdialysis [11] and appears present in patients with
reduced GFR and no metabolic acidosis detected with indirect
techniques [26]. Although patients with chronically reduced
GFR, like Nx, can mount the same UNAE patients with intact
nephron mass in the steady state [27, 28], their initial increase
in UNAE in response to an increment in dietary H+ is less than
the increase in intrinsic H+ production [29]. These patients
eventually achieve H+ excretion equivalent to intrinsic H+ production [28] and can then maintain normal plasma acid–base
parameters despite reduced GFR [30]. We hypothesize that
patients develop H+ retention during the interval when UNAE
is less than intrinsic H+ production. Animal studies from our
laboratory support that this H+ retention persists as long as
the increment in dietary H+ continues and remits only after
the increment in dietary H+ is stopped [31]. Consequently, we
hypothesize that patients with GFR that is reduced but not
associated with metabolic acidosis who eat the high H+ diets
of industrialized societies [32] are able to balance urine H+
excretion with H+ intake but do so in a setting of H+ retention
[26]. Consequently, H+ retention with or without metabolic
acidosis might contribute to progressive GFR decline in
patients with reduced GFR.
Because H+ retention occurred in these 2/3 Nx animals with
plasma acid–base parameters not different from Sham, plasma
appears not to be a fluid compartment containing the excess
free H+. Other investigators using microdialysis report that the
microdialyzate interfaces with interstitial fluid [33]. We hypothesize that interstitial fluid is at least one compartment containing the excess free H+. With comparatively little protein, H+
additions to interstitial fluid would cause rapid and large [H+]
changes compared with plasma because of its relative lack of
buffers. We further hypothesize that these measured [H+]
changes signal, among other kidney responses, increased kidney
levels of AII, endothelin and aldosterone, each of which contributes to enhance nephron acid excretion [8] and to GFR decline
as supported by previous [12] and the present studies. Furthermore, we hypothesize that when the interstitial fluid H+ capacity
is neared with continued H+ addition to body fluids, excess H+
entering plasma exceeds capacity of plasma and blood buffers
to take on additional H+ without a reduction in pH and
[HCO3]. We believe that the lower plasma pH and [HCO3]
with unchanged H+ retention in Nx with valsartan compared
with Nx without valsartan (Tables 2 and 3) reflects the movement of additional accumulated H+ into plasma in the setting
of valsartan inhibition of superficial distal nephron acidification
shown in earlier studies [8].
Patients with reduced GFR have increased urine excretion
of angiotensinogen [34], a marker of kidney AII levels [35],
and oral NaHCO3 reduces urine excretion of angiotensinogen
in patients with reduced GFR [36]. These data suggest that H+
retention increases and its amelioration with alkali salts decreases kidney AII in patients with reduced GFR, like in Nx of
previous [8] and the present studies. In addition, dietary H+
reduction with alkali salts is an effective adjunct to angiotensin-converting enzyme inhibition to help preserve GFR in
CKD patients with [36–38] and without [9] metabolic acidosis. Thus, alkali salts might synergistically reduce kidney AII
effect when combined with anti-AII drugs.
D. E. Wesson et al.
In summary, the present study used 2/3 Nx to model patients with reduced GFR and progressive GFR decline despite
no metabolic acidosis to show that H+ retention mediated
GFR decline through AII receptors. Dietary NaHCO3 but not
AII receptor antagonism reduced H+ retention and NaHCO3
conserved GFR better than AII receptor antagonism and
better than combined AII, endothelin A and aldosterone receptor antagonism. These studies support continued testing
the comparatively inexpensive, safe and well-tolerated intervention of dietary HCO3 as an adjunct to blood pressure reduction and anti-AII pharmacologic therapy to preserve GFR
in patients with reduced GFR at presentation.
AC K N O W L E D G E M E N T S
We are grateful to Callenda Hacker, Geraldine Tasby and
Cathy Hudson for expert technical assistance. The study was
supported by funds from the University Medical Center
(Lubbock, TX) Endowment and the Larry and Jane Woirhaye
Memorial Endowment in Renal Research at the Texas Tech
University Health Sciences Center.
The results of this paper have not been published previously in
whole or in part, except in abstract form.
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Received for publication: 9.7.2014; Accepted in revised form: 22.11.2014
Nephrol Dial Transplant (2015) 30: 770–781
doi: 10.1093/ndt/gfu384
Advance Access publication 18 December 2014
Impaired expression of key molecules of ammoniagenesis
underlies renal acidosis in a rat model of chronic kidney disease
Remy Bürki1,*, Nilufar Mohebbi1,2,*, Carla Bettoni1, Xueqi Wang2,3, Andreas L. Serra2
and Carsten A. Wagner1
Institute of Physiology and ZIHP, University of Zurich, Zurich, Switzerland, 2Division of Nephrology, University Hospital Zurich, Zurich,
ORIGINAL ARTICLE
1
Switzerland and 3Department of Nephrology, Changzheng Hospital, Second Military Medical University, Shanghai, China
Correspondence and offprint requests to: Carsten A. Wagner; E-mail: [email protected]
*These authors have contributed equally to this work and therefore share first authorship.
low urinary pH. In contrast, the RhCG ammonia transporter,
critical for the final secretion of ammonia into urine was
strongly down-regulated in CKD animals.
Conclusion. In the Han:SPRD rat model for CKD, key molecules required for renal ammoniagenesis and ammonia excretion are highly down-regulated providing a possible molecular
explanation for the development and maintenance of renal
acidosis in CKD patients.
A B S T R AC T
Background. Advanced chronic kidney disease (CKD) is associated with the development of renal metabolic acidosis. Metabolic acidosis per se may represent a trigger for progression of
CKD. Renal acidosis of CKD is characterized by low urinary
ammonium excretion with preserved urinary acidification indicating a defect in renal ammoniagenesis, ammonia excretion
or both. The underlying molecular mechanisms, however,
have not been addressed to date.
Methods. We examined the Han:SPRD rat model and used a
combination of metabolic studies, mRNA and protein analysis
of renal molecules involved in acid–base handling.
Results. We demonstrate that rats with reduced kidney
function as evident from lower creatinine clearance, lower
haematocrit, higher plasma blood urea nitrogen, creatinine,
phosphate and potassium had metabolic acidosis that could be
aggravated by HCl acid loading. Urinary ammonium excretion
was highly reduced whereas urinary pH was more acidic in
CKD compared with control animals. The abundance of key
enzymes and transporters of proximal tubular ammoniagenesis ( phosphate-dependent glutaminase, PEPCK and SNAT3)
and bicarbonate transport (NBCe1) was reduced in CKD compared with control animals. In the collecting duct, normal expression of the B1 H+-ATPase subunit is in agreement with
© The Author 2014. Published by Oxford University Press
on behalf of ERA-EDTA. All rights reserved.
Keywords: acidosis, ammoniagenesis, CKD
INTRODUCTION
Metabolic acidosis is common in chronic kidney disease (CKD)
and is associated with several complications such as muscle
wasting, impaired growth in children, bone disease, hypoalbuminaemia, inflammation and insulin resistance. Moreover,
it has been associated with increased mortality in dialysis and
non-dialysis-dependent CKD patients [1–5]. Numerous recent
studies particularly highlighted the role of metabolic acidosis in
the progression of CKD and the increased risk to develop endstage renal disease [6–9]. The mechanisms of metabolic acidosis
in CKD were first functionally investigated >50 years ago and it
was shown that patients with metabolic acidosis and CKD demonstrate reduced excretion of ammonium, the most important
770