Physiol Genomics 40: 158–166, 2010.
First published November 17, 2009; doi:10.1152/physiolgenomics.00088.2009.
Transcriptional and physiological responses to chronic ACTH treatment by
the mouse kidney
Donald R. Dunbar, Hiba Khaled, Louise C. Evans, Emad A. S. Al-Dujaili, Linda J. Mullins,
John J. Mullins, Christopher J. Kenyon, and Matthew A. Bailey
Centre for Cardiovascular Science, Queen’s Medical Research Institute, University of Edinburgh, Edinburgh,
United Kingdom
Submitted 28 May 2009; accepted in final form 17 November 2009
Dunbar DR, Khaled H, Evans LC, Al-Dujaili EA, Mullins LJ,
Mullins JJ, Kenyon CJ, Bailey MA. Transcriptional and physiolog-
ical responses to chronic ACTH treatment by the mouse kidney.
Physiol Genomics 40: 158 –166, 2010. First published November 17,
2009; doi:10.1152/physiolgenomics.00088.2009.—We investigated
the effects on urinary steroid and electrolyte excretion and renal gene
expression of chronic infusions of ACTH in the mouse. ACTH caused
a sustained increase in corticosteroid excretion; aldosterone excretion
was only transiently elevated. There was an increase in the excretion
of deoxycorticosterone, a weak mineralocorticoid, to levels of phys-
iological significance. Nevertheless, we observed neither antinatriure-
sis nor kaliuresis in ACTH-treated mice, and plasma renin activity
was not suppressed. We identified no changes in expression of
mineralocorticoid target genes. Water turnover was increased in
chronic ACTH-treated mice, as were hematocrit and hypertonicity:
volume contraction is consistent with high levels of glucocorticoid.
ACTH-treated mice exhibited other signs of glucocorticoid excess,
such as enhanced weight gain and involution of the thymus. We
identified novel ACTH-induced changes in 1) genes involved in
vitamin D (Cyp27b1, Cyp24a1, Gc) and calcium (Sgk, Calb1, Trpv5)
metabolism associated with calciuria and phosphaturia; 2) genes that
would be predicted to desensitize the kidney to glucocorticoid action
(Nr3c1, Hsd11b1, Fkbp5); and 3) genes encoding transporters of
enzyme systems associated with xenobiotic metabolism and oxidative
stress. Although there is evidence that ACTH-induced hypertension is
a function of physiological cross talk between glucocorticoids and
mineralocorticoids, the present study suggests that the major changes
in electrolyte and fluid homeostasis and renal function are attributable
to glucocorticoids. The calcium and organic anion metabolism path-
ways that are affected by ACTH may explain some of the known
adverse effects associated with glucocorticoid excess.
renal function; gene expression; adrenocorticotropic hormone; cal-
cium; steroids
THERE ARE VARIOUS PATHOLOGICAL, genetic, and environmental
circumstances that directly or indirectly lead to activation of
the hypothalamo-pituitary-adrenal (HPA) axis and thereby
cause an increase in blood pressure. Patients with Cushing
syndrome and those exposed to chronic stress exemplify this
process: ACTH causes concomitant adrenal gland hypertrophy,
adrenocorticosteroid production, and hypertension. Studies of
the etiology of the causes of HPA-mediated blood pressure
increases have focused generally on the mineralocorticoid and
glucocorticoid properties of adrenal secretions, and although
very many contributory factors have been implicated (27, 45),
no clear-cut mechanism has emerged. Other cardiovascular
Address for reprint requests and other correspondence: D. R. Dunbar, Centre
for Cardiovascular Science, Queen’s Medical Research Inst., Univ. of Edin-
burgh, Edinburgh EH16 4TJ, UK (e-mail: donald.dunbar@ed.ac.uk).
158 1094-8341/10 $8.00 Copyright © 2010 the American Physiological Society
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risk factors including obesity, dyslipidemia, and diabetes are
also associated with glucocorticoid excess.
Increased mineralocorticoid activity might be expected for
three reasons. First, secretion of the principal mineralocorti-
coid, aldosterone, in the zona glomerulosa is driven in part by
ACTH (46). Second, intermediates in corticosteroid biosyn-
thetic pathways (e.g., deoxycorticosterone) are also stimulated
by ACTH, and these also have mineralocorticoid properties
(23, 42). Third, endogenous glucocorticoids (e.g., cortisol and
corticosterone) are also potential agonists of mineralocorticoid
receptors (MR), particularly when circulating levels are high
enough to overcome the protective 11-oxidation step that
inactivates glucocorticoid hormones in MR target tissues (39).
Arguably, long-term blood pressure is determined primarily
by the kidney. Mineralocorticoids are associated with sodium
and water reabsorption by the distal tubule, leading to volume
expansion at least in the short to medium term. Glucocorticoids
also influence renal function independent of MR via glucocor-
ticoid receptors (GR): GR-specific ligands will affect glomer-
ular filtration rate (GFR) and proximal tubular function (3, 37),
and GR activation may also influence sodium transport in the
distal nephron (16). In the case of HPA activation we have
shown (6) that both MR and GR processes can increase
epithelial sodium channel (ENaC) activity.
In the present study we have employed chronic ACTH
treatment in mice as a model of HPA-mediated hypertension,
defining a time course for altered steroidogenesis and electro-
lyte metabolism. In combination with a microarray analysis of
renal gene expression, we have further resolved the responses
to ACTH excess into MR- and GR-mediated components. We
have identified novel pathways, validated physiologically, that
may explain some of the pathophysiological consequences of
HPA hypertension.
MATERIALS AND METHODS
ACTH treatment. Groups of age-matched male C57BL/6 mice
(Harlan Olac) weighing ⬃25 g were fed a diet containing 0.3% Na
(SDS Diets, Witham, UK) with free access to water in a temperature-
and light-controlled (12:12-h light-dark cycle) room. On day 0 an
osmotic minipump (model 2002, Alzet, Charles River) was implanted
subcutaneously under halothane anesthesia. Pumps contained either
ACTH (Synacthen, Ciba-Geigy; 2.8 ␮g/day) or vehicle (0.9% NaCl).
Animal studies were undertaken under UK Home Office license,
following review by the local ethics committee. All experiments were
performed in accordance with the UK Home Office Animals (Scien-
tific Procedures) Act of 1986.
Metabolic cage studies. For metabolic studies, mice were housed
individually in cages (model 3600M021, Techniplast) with free access
to food and water as described above. After a 4-day period of
acclimatization, daily food and fluid intakes and body weights were
 RENAL EFFECTS OF CHRONIC ACTH TREATMENT IN MICE 159
recorded and urine samples were collected for later analysis. Mea- t-test or ANOVA with Bonferroni post hoc test. For cumulative
surements were made for three consecutive days to obtain baseline balance data, box plots are presented and medians are compared with
recordings. Food and water intake and excretion of steroids and the Mann-Whitney test.
electrolytes were stable during this period and were therefore aver-
aged to generate a single baseline value (0 in Figs. 1–3). Experimental RESULTS
measurements were then made for 12 further days (days 1–12 in Figs.
1–3) after minipump implant. Mice received either ACTH (n ⫽ 10) or Adrenocortical function. Adrenal mass and plasma cortico-
saline vehicle (n ⫽ 10). In a subset of animals, GFR was measured sterone were increased threefold by ACTH treatment (Table 1).
from the clearance of FITC-inulin, as described previously (6). Urinary corticosterone values started to increase after 1 day of
Tissue collection. Further cohorts of ACTH- and vehicle-treated ACTH treatment (Fig. 1A) and continued to rise, reaching a
mice were prepared for tissue sampling and blood pressure measure- plateau at day 10, when rates of excretion were ⬃65-fold
ments. Mice were housed in pairs, and blood pressure was measured higher than in vehicle-treated control mice (P ⬍ 0.001). Uri-
by tail-cuff plethysmography as previously described (14). Mice were
killed by CO2 inhalation after restraint stress, maximizing plasma
nary aldosterone also increased initially, reaching a peak that
corticosterone levels. Heparinized blood was collected by cardiac was 9.6-fold higher than in control mice at day 1 (Fig. 1B).
puncture, and plasma was stored for measurement of electrolytes, Thereafter, levels declined and had returned to pretreatment
osmolality (freezing point depression), and hormones (32). values by day 10. Urinary deoxycorticosterone followed a
Urinalysis. Urinary sodium, potassium, and calcium were mea- pattern similar to that of corticosterone and at the end of the
sured by flame photometry (BWB Technologies UK). Phosphate was ACTH treatment period was 80-fold higher than control mice
measured by a colorimetric method (QuantiChrom DIPI500, Bio- (Fig. 1C). It is notable that in a previous study of a mouse
Assay Systems). Urinary aldosterone, deoxycorticosterone, and cor- model of congenital adrenal hyperplasia, hypertension was
ticosterone were measured by specific ELISAs (1, 2). The effects of observed with similar high levels of urinary deoxycorticoste-
ACTH on urinary aldosterone levels were used to validate an ELISA rone when both urinary corticosterone and aldosterone were
for mouse urine and have been published previously (1).
reduced (32).
Creatinine in urine was determined with the creatininase/creati-
nase-specific enzymatic method (8) and a commercial kit (Alpha Water and electrolyte balance. Urine flow rate was in-
Laboratories UK) adapted for use on a Cobas Fara centrifugal ana- creased almost from the start of treatment (Fig. 2A) and was
lyzer (Roche Diagnostics). Within-run precision was coefficient of matched by increased drinking (data not shown). By the second
variation (CV) ⬍ 3%, while intrabatch precision was CV ⬍ 5%. week of treatment, however, the polydipsia exceeded the poly-
Mouse albumin measurements were determined with a commercial uria and ACTH-treated mice showed a significantly more
microalbumin kit (Olympus Diagnostics) adapted for use on a Cobas positive cumulative water balance (Fig. 2B). Antinatriuresis
Fara centrifugal analyzer. The immunoturbidimetric assay was stan- was observed on the first experimental day in both ACTH- and
dardized against purified mouse albumin standards (Sigma Chemical vehicle-treated mice (Fig. 2C). This probably relates to the
UK), with samples diluted in phosphate-buffered saline as appropri- effects of surgery, since food (and therefore sodium) intake
ate.
during the first 24 h was similarly reduced. By day 2 of
RNA preparation and microarray processing. Kidney RNA for
microarray analysis was prepared by the TRIzol method and then treatment, food intake had recovered to preoperative levels in
processed through standard Affymetrix protocols, with one round of both groups of mice and was not thereafter affected by ACTH.
cDNA amplification. Processed RNAs were hybridized to the Af- Control mice maintained a neutral sodium balance throughout
fymetrix Mouse Genome 430 2.0 GeneChip. RNA processing and the experiment. In contrast, ACTH treatment induced a signif-
microarray analyses were carried out by ARK Genomics (Roslin icant natriuresis. Indeed, sodium excretion exceeded intake
Institute, Edinburgh, UK). Data were extracted through the GCOS
software, and CEL files were used for further data processing. CEL
files were imported into Bioconductor (18), normalized by RMA in Table 1. Body weight, organ weight, blood pressure, and
the Affy (17) module, and statistically analyzed with the Limma (41) plasma metabolite data for C57BL/6J mice treated with
package and also with the Rank Products (RankProd) package (9). vehicle or ACTH
Gene Ontology (4) and KEGG pathway enrichment analysis was done
with the Webgestalt tool (49). Microarray data are available in the Vehicle P Value ACTH
ArrayExpress database (www.ebi.ac.uk/arrayexpress) under accession PNa, mmol/l 142.6 ⫾ 1.0 ⬍0.01 147.9 ⫾ 1.5
number E-MEXP-1761. PK, mmol/l 4.79 ⫾ 0.23 ⬍0.05 3.86 ⫾ 0.27
Real-time RT-PCR. Selected genes were quantified by preopti- POsm, mosmol/kgH2O 306 ⫾ 1 ⬍0.01 316 ⫾ 2
mized RT-PCR assays in kidneys from separate cohorts of vehicle- Hematocrit, % 42.4 ⫾ 1.1 ⬍0.001 48.2 ⫾ 1.1
and ACTH-treated mice. Total RNA was extracted from tissue sam- Pcort, nmol/l 133 ⫾ 29 ⬍0.01 745 ⫾ 124
ples with the Qiagen RNeasy system and reverse transcribed into PRA, ng·ml⫺1·h⫺1 11.6 ⫾ 2.2 NS 8.0 ⫾ 0.88
cDNA with random primers and the QuantiTect DNase/reverse tran- PRC, ng·ml⫺1·h⫺1 1,520 ⫾ 210 NS 1,991 ⫾ 282
AGT, ng·ml⫺1·h⫺1 304 ⫾ 23 NS 312 ⫾ 20
scription kit (Qiagen, Crawley, UK). cDNA (equivalent to 1 ng total
⌬Body wt, g 1.5 ⫾ 0.25 ⬍0.05 2.36 ⫾ 0.38
RNA) was incubated in triplicate with gene-specific primers and Heart wt, mg/g body wt 6.39 ⫾ 0.25 NS 5.98 ⫾ 0.24
fluorescent probes (using predesigned assays from Applied Biosys- Adrenal wt, mg/g body wt 0.11 ⫾ 0.1 ⬍0.001 0.28 ⫾ 0.03
tems, Warrington, UK) in 1⫻ Roche LightCyclerR480 Probes mas- Thymus wt, mg/g body wt 1.75 ⫾ 0.11 ⬍0.001 0.63 ⫾ 0.11
termix. PCR cycling and detection of fluorescent signal was carried Systolic BP, mmHg 104.8 ⫾ 2.4 ⬍0.01 127.8 ⫾ 2.9
out with a Roche LightCyclerR480. A standard curve was constructed GFR, ml/min 0.43 ⫾ 0.08 ⬍0.05 0.62 ⫾ 0.01
for each primer probe set with a serial dilution of cDNA pooled from
Data are means ⫾ SE for C57BL/6J mice treated with either vehicle (n ⫽ 8) or
all samples. Results were corrected for the mean of expression of ACTH (n ⫽ 8) for 14 days. PNa, plasma sodium; PK, plasma potassium; POsm,
␤-actin and 18S ribosomal RNA. Neither 18S RNA nor ␤-actin was plasma osmolality; Pcort, plasma corticosterone; PRA, plasma renin activity; PRC,
affected by ACTH treatment. plasma renin concentration; AGT, angiotensinogen; ⌬Body wt, change in body
Statistical analyses. Data are presented as means ⫾ SE. After tests weight; BP, blood pressure; GFR, glomerular filtration rate. Statistical compari-
for Gaussian distribution, comparisons were made by either unpaired sons were made by t-test, and P values are as given; NS, not significant.

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160 RENAL EFFECTS OF CHRONIC ACTH TREATMENT IN MICE
Fig. 1. Daily urinary excretion of corticosterone (A), aldosterone (B), and
deoxycorticosterone (C). Values are means ⫾ SE; n ⫽ 5 (ACTH, Œ) and 6
(vehicle,
) except for deoxcorticosterone control values, where n ⫽ 3
because of sample insufficiency.
from day 3 onward, and ACTH-treated mice were in a negative
sodium balance during both the first and second weeks (Fig.
2D). ACTH did not affect urinary potassium excretion (data
not shown), but these mice were nevertheless mildly hypokale-
mic (Table 1). The urinary Na-to-K concentration ratio was
thus significantly higher in ACTH-treated mice than in control
mice (Fig. 2E). Although ACTH-treated mice were in a posi-
tive water balance, there was no evidence for expansion of
plasma volume. Indeed, hematocrit was significantly higher in
mice receiving ACTH (Table 1): the hypernatremia and in-
creased plasma osmolality may therefore reflect the concen-
trating effects of a reduction in plasma volume. Systolic blood
pressure was significantly increased (Table 1). Chronic ACTH
treatment increased GFR (Table 1) and induced the develop-
ment of albuminuria (Fig. 2F), particularly during the second
phase of the experimental regimen.
Organ weights. Body and organ weight data are summarized
in Table 1. Thymus weights were reduced by ACTH, in
keeping with modulatory effects of excess glucocorticoid ac-
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tivity. Heart and kidney were unaffected, and liver was slightly
increased. Body weight gain across the experimental cohorts
was consistently greater in ACTH-treated mice than in control
mice. In mice used for microarray studies, those treated with
ACTH showed a significant gain compared with vehicle-
treated control mice; in contrast, there were no significant
differences in animals placed in metabolic cages.
Microarray analysis. Gene expression analysis revealed a
relatively small degree of differential gene expression between
control and ACTH-treated mice, in terms of both numbers of
genes and the extent of modulation. Only 186 probe sets were
differentially expressed by twofold or more (Supplemental
Table S1).1 Only 12 probe sets were differentially expressed by
5-fold or more, and only 3 exceeded 10-fold. After accounting
for redundant probe sets (probe sets mapping to the same gene
annotation), this corresponded to 85 genes at least twofold
upregulated by ACTH and 64 genes similarly downregulated.
Gene function (Gene Ontology and KEGG pathway) enrich-
ment analysis revealed several biological pathways that were
statistically significantly affected in the up- and downregulated
gene sets. Among these pathways were those involved in
glucocorticoid hormone signaling, calcium homeostasis, and
xenobiotic metabolism. To validate the expression data from
the microarray studies, quantitative real-time RT-PCR analysis
was carried out on selected genes (Table 2). Parametric
(Limma) and nonparametric (Rank Products) statistical tests
gave results for selection of differential expressed gene lists
similar to the fold change criteria.
Mineralocorticoid-related genes. The effects of ACTH on
genes implicated in mineralocorticoid actions are shown in
Table 3. Sgk1, which mediates mineralocorticoid effects on ion
channel activity, and Scnn1a (amiloride-sensitive ENac, ␣-sub-
unit) were upregulated (4.4- and 1.7-fold, respectively), al-
though the effects were not statistically significant after mi-
croarray multiple testing correction. The expression of the
other ENaC subunits was not altered. Similarly, Kras, Nedd4,
and Fxyd4, which are also implicated in mineralocorticoid
signaling, were not changed. Mineralocorticoid (but not glu-
cocorticoid)-induced hypertension is usually associated with
renin (Ren) suppression. Neither renin gene expression levels
nor plasma renin levels were affected, although angiotensin-
converting enzyme (Ace) was markedly suppressed. The lack
of effect of ACTH on Ren expression was confirmed by
RT-PCR (Table 2). MR (Nr3c2) gene expression was not
affected. The apelin receptor (Agtrl1) was significantly down-
regulated by ACTH treatment. This may be relevant to blood
pressure control since apelin is a vasoactive peptide. In the
kidney, the apelin receptor is expressed in all nephron seg-
ments, with highest expression in the glomerulus (21). Apelin
has complex effects on the pre- and postglomerular microvas-
culature regulating renal hemodynamics (21), and the physio-
logical impact of decreased receptor expression during ACTH
excess is unclear.
Glucocorticoid-related genes. Glucocorticoid response ele-
ments are found in the promoters of a large number of genes;
those in Table 3 were selected on the basis of associations with
glucocorticoid signaling, with well-established metabolic ef-
fects of glucocorticoids, or with previously identified renal
1
The online version of this article contains supplemental material.
 RENAL EFFECTS OF CHRONIC ACTH TREATMENT IN MICE 161

Fig. 2. A: daily urine flow rate in mice receiving either ACTH (; n ⫽ 5) or saline vehicle (□; n ⫽ 6) for 12 days. Baseline excretion (day 0) is the mean of
measurements obtained on 3 consecutive days before implant of the osmotic minipump. B: cumulative sodium balance for days 1– 6 and 7–12 of treatment. Open
bars, vehicle; filled bars, ACTH. C: daily sodium excretion in mice receiving ACTH () or saline vehicle (□). D: cumulative sodium balance for days 1– 6 and
7–12 of treatment. Open bars, vehicle; filled bars, ACTH. E: urinary sodium-to-potassium concentration ratio. □, Vehicle; , ACTH. F: urinary
albumin-creatinine ratio. Open bars, vehicle; filled bars, ACTH. For A, C, E, and F, data are means ⫾ SE. Statistical comparisons were made with 2-way
ANOVA, which showed in both cases a highly significant effect of treatment (P ⬍ 0.001). Post hoc comparisons were made with Bonferroni test. *P ⬍ 0.05;
**P ⬍ 0.01. For B and D, medians and ranges are shown and comparisons were made with the Mann-Whitney test.

target genes. GR (Nr3c1) and hydroxysteroid 11␤-dehydroge-
nase 1 (Hsd11b1) expression were downregulated, possibly to
mitigate the effects of excess hormone. Similarly, Fkbp5, an
immunophilin protein that affects GR trafficking, lowers ago-
nist binding affinity, and causes glucocorticoid resistance, was
upregulated more than twofold. Agt, Pck1, and Tat (not shown)
are classical GR-regulated genes but were not affected by
ACTH. Of the glucocorticoid-inducible genes that have been
implicated in the regulation of renal function, only the chloride
channel Ka (Clcnka) was significantly affected by ACTH.
Calcium homeostasis. Of the 85 genes upregulated by
greater than twofold, 5 were annotated in the Gene Ontology
(GO) as being involved in calcium homeostasis and the regu-
lation of vitamin D activity (GO: 0006766 and GO: 0019842;
also see Table 3). Sgk1 has been shown to control expression
of calbindin-28K (Calb1), which binds and transports Ca2⫹
from apical to basolateral surfaces of epithelial cells. Calb1 is
thought to act in concert with an apical membrane calcium
channel (Trpv5) and a basolateral Na/Ca exchanger (Slc8a1) to
promote calcium reabsorption in epithelial cells of the distal
tubule. Accordingly, expression of these three genes was quan-
tified by RT-PCR: increased expression of Calb1 was con-
firmed, Slc8a1 was unaffected (as it was in the microarray
analysis), and Trpv5 (not represented on the microarray) was
down- rather than upregulated.
Vitamin D also transcriptionally regulates Trpv5 (20), and, in
the microarray, three genes controlling vitamin D activity were
affected: cytochrome P-450, family 27, subfamily b, polypeptide
1 (Cyp27b1) encoding the 1␣-hydroxylase enzyme responsible
for the final step in the synthesis of the hormonally active 1,25-
dihydroxyvitamin D; cytochrome P-450, family 24, subfamily a,
polypeptide 1 (Cyp24a1) encoding the deactivating 24-hydroxy-
lase enzyme; and group-specific component (Gc), otherwise
known as vitamin D binding protein. Real-time PCR confirmed
the magnitude of Cyp24a1 changes, but the effect was not statis-
tically significant, as with the microarray data.

Table 2. RT-PCR data for selected genes

Symbol Gene Vehicle P Value ACTH

Fkbp5 FK 506 binding protein 5 42 ⫾ 7 0.01 196 ⫾ 52

Aqp4 Aquaporin 4 43 ⫾ 8 0.05 104 ⫾ 22
Cyp2b10 Cytochrome P-450 family 2 subfamily b, polypeptide 10 6⫾6 0.01 280 ⫾ 116
Ren1 Renin 422 ⫾ 158 0.81 775 ⫾ 412
Calb1 Calbindin-28K 34 ⫾ 4 0.05 68 ⫾ 16
Trpv5 Transient receptor potential cation channel, subfamily V 109 ⫾ 11 0.05 67 ⫾ 12
Cyp241a Cytochrome P-450 family 24 subfamily b, polypeptide 1 284 ⫾ 93 0.5 595 ⫾ 299
Slc8a1 Solute carrier family 8a member 1 49 ⫾ 7 0.4 65 ⫾ 11
P values are for the t-test with biological replicates.

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162 RENAL EFFECTS OF CHRONIC ACTH TREATMENT IN MICE

Table 3. Microarray gene expression data for selected genes

Symbol Gene Vehicle P Value ACTH

Mineralocorticoid-related genes
Ren1 Renin 235 ⫾ 29 0.97 295 ⫾ 127
Ace Angiotensin-converting enzyme 360 ⫾ 21 0.11 160 ⫾ 29
Agtrl1 Angiotensin receptor-like 1 (Apelin receptor) 122 ⫾ 17 ⬍0.01 15 ⫾ 3
Sgk1 Serum/glucocorticoid-regulated kinase 309 ⫾ 78 0.1 1,292 ⫾ 229
Nr3c2 Mineralocorticoid receptor 29.7 ⫹ 0.9 0.94 28.7 ⫾ 0.7
Kras Kirsten-Ras 291 ⫾ 1 0.94 307 ⫾ 6
Sccn1 Epithelial sodium channel ␣ 218 ⫾ 19 0.077 330 ⫾ 29
Nedd4 Neural precursor cell expressed, developmentally downregulated 4 984 ⫾ 40 0.99 992 ⫾ 26
Fxyd4 Fxyd containing ion transport regulator 4 674 ⫾ 50 0.66 556 ⫾ 22
Glucocorticoid-related genes
Nr3c1 Glucocorticoid receptor 442 ⫾ 22 0.27 332 ⫾ 24
Hsd11b1 11␤-Hydroxysteroid dehydrogenase type 1 1,759 ⫾ 248 0.04 654 ⫾ 59
Hsd11b2 11␤-Hydroxysteroid dehydrogenase type 2 570 ⫾ 28 0.85 616 ⫾ 39
Fkbp5 FK 506 binding protein 5 382 ⫹ 33 0.02 1,024 ⫾ 70
Pck1 Phosphoenolpyruvate carboxykinase 1 2,950 ⫾ 335 0.45 4,444 ⫾ 685
Agt Angiotensinogen 257 ⫾ 13 0.21 431 ⫾ 67
Atpa1 Na-K-ATPase 4,188 ⫾ 117 0.43 5,274 ⫾ 430
Slc12a1 Sodium potassium chloride cotransporter 2,937 ⫾ 530 0.91 3,116 ⫾ 151
Clcnka Chloride channel Ka 271 ⫾ 6 0.05 460 ⫾ 33
Kcnj1 ROMK channel 1,024 ⫾ 94 0.55 1,240 ⫾ 50
Calcium regulation-related genes
Calb1 Calbindin-28K 3,238 ⫾ 285 0.04 6,332 ⫾ 332
Cyp27b1 Cytochrome P-450 family 27 subfamily b, polypeptide 1 18 ⫾ 5 0.14 136 ⫾ 65
Cyp24a1 Cytochrome P-450 family 24 subfamily b, polypeptide 1 472 ⫾ 163 0.43 1,076 ⫾ 182
Gc Group-specific component 63 ⫾ 6 0.47 148 ⫾ 60
Sgk Serum/glucocorticoid-regulated kinase 309 ⫾ 78 0.1 1,292 ⫾ 229
Organic ion transporters and metabolizing enzyme genes
Cyp2b10 Cytochrome P-450 family 2 subfamily b, polypeptide 10 23 ⫾ 4 0.22 317 ⫾ 104
Cyp4a14 Cytochrome P-450 family 4 subfamily a, polypeptide 14 350 ⫾ 233 0.43 990 ⫾ 374
Aldh1a1 Aldehyde dehydrogenase family 1, subfamily a1 45 ⫾ 9 0.19 149 ⫾ 40
Aldh1a7 Aldehyde dehydrogenase family 1, subfamily a7 137 ⫹ 17 0.16 378 ⫾ 91
Gpx6 Glutathione peroxidase 6 202 ⫾ 27 0.12 409 ⫾ 52
Ttpat Tocopherol (␣) transfer protein 151 ⫾ 11 0.09 395 ⫾ 83
Sult1a1 Sulfotransferase family 1a phenol preferring, member 1 163 ⫾ 15 0.02 488 ⫾ 66
Slc22a7 Solute carrier family 22a member 7 1,702 ⫾ 29 0.22 828 ⫾ 246
Slc22a8 Solute carrier family 22a member 8 2,510 ⫾ 266 0.01 900 ⫾ 39
Slco1a1 Solute carrier organic anion transporter, member 1a1 2,381 ⫾ 260 0.22 634 ⫾ 261
P values are corrected for multiple testing.

The mixed response of genes regulating renal calcium ex-
cretion precludes a coherent predictive model of renal calcium
handling. We therefore measured urinary calcium and phos-
phate excretion directly, finding both to be increased strikingly
by ACTH (Fig. 3). Notably, genes involved in renal phosphate
reabsorption (e.g., Npt2) were not differentially expressed on
the microarray.
Transporters and exchangers. Slco1b1, Slc22a7, and Slc22a8
are expressed on basolateral membranes of proximal tubular
cells and are thought to have a role in organic ion excretion.
The expression of all three was decreased by ACTH (Table 3).
A compensatory downregulation of excretion might be stimu-
lated by the need to retain endogenous molecules either in the
face of the catabolic effects of glucocorticoids or to offset the
increased glomerular filtration induced by ACTH (6). It is
notable that the renal expression of ABC transporters (gener-
ally involved in reabsorption of chemicals at the basolateral
membrane) was unaffected. The expression of Ttpa [tocoph-
erol (␣) transfer protein] was increased twofold by ACTH.
This protein mobilizes the antioxidant vitamin E into the
circulation and may therefore be induced to limit the oxi-
dative stress effects of glucocorticoid excess. Likewise,
glutathione peroxidase activity also protects against oxida-
tive damage by reducing lipid and hydrogen peroxides to
alcohols and water. However, a role for the peroxidase
isozyme encoded by Gpx6, the gene regulated by ACTH,
has yet to be established.
The remaining genes in Table 3 encode enzymes involved in
xenobiotic metabolism that prototypically are induced by dexa-
methasone. It is likely, therefore, that they are responding to
excess glucocorticoids in the present experiment. Most are
involved in lipid metabolism. Cyp4a14 changes are of partic-
ular interest since the enzyme catalyzes the conversion of
arachidonic acid to a prohypertensive compound, 20-hydrox-
yarachidonic acid, and may also promote steroid hormone
clearance. Sult1a1 is a glucocorticoid-inducible sulfotrans-
ferase enzyme that potentially stimulates steroid clearance by
conversion to water-soluble sulfate metabolites.
Other interesting genes. A complete list of genes regulated
by ACTH is available in Supplemental Table S1. Among the

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 RENAL EFFECTS OF CHRONIC ACTH TREATMENT IN MICE
Fig. 3. Twenty-four-hour urinary excretion of calcium (A) and phosphate (B)
in mice receiving either ACTH (; n ⫽ 5) or saline vehicle (□; n ⫽ 6) for 12
days. Baseline excretion (day 0) is the mean of measurements obtained on 3
consecutive days before implant of the osmotic minipump. Data are means ⫾
SE. Statistical comparisons were made with 2-way ANOVA, which showed in
all cases a highly significant effect of treatment (P ⬍ 0.001). Post hoc
comparisons were made with Bonferroni test. *P ⬍ 0.05; **P ⬍ 0.01.
upregulated genes, those that are potentially relevant to car-
diovascular risk are ApoCIII and Angptl. These are inhibitors
of lipoprotein lipase (Lpl), which is itself downregulated. The
transporters Slc6a15 (a chloride-dependent amino acid neuro-
transmitter transporter) and Slc9a8 (NHE8) were downregu-
lated.
DISCUSSION
The metabolic and renovascular effects of murine ACTH
excess are of particular interest because of an expanding list of
genetically modified mouse strains in which metabolic disease
can be linked to activation of the HPA axis (7, 31, 32). In the
present study plasma aldosterone and corticosterone were ini-
tially both stimulated by ACTH, but the effect on aldosterone
was transient (34) and only the effect on corticosterone was
sustained. Persistent glucocorticoid excess implicates GR-me-
diated pathways in the ACTH phenotype, but MR-mediated
responses may also be physiologically important for two rea-
sons. First, corticosterone will bind MR if concentrations are
high enough to overcome the protective barrier of Hsd11b2
(25). Second, deoxycorticosterone, a weak MR agonist, was
increased to a level of cardiovascular significance (32).
We anticipated that microarray analysis would define the
effect of chronic ACTH on renal gene expression, and help to
delineate between MR- and GR-mediated pathways. These
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163
studies have two major limitations. First, MR- and GR-medi-
ated changes in gene expression often converge on the same
pathways, and we are not able to unequivocally attribute
changes in gene expression to a given receptor. This is exem-
plified by the more than fourfold increase in Sgk1, which is
under the transcriptional control of both MR and GR (26).
Second, relatively few cells within the kidney express MR, and
it is possible that whole kidney analysis is insufficiently sen-
sitive to detect physiologically significant changes in the ex-
pression of target genes. We were, for example, unable to
detect significant changes in the expression of key sodium
transport proteins such as ␣-ENaC (Scnn1a; 1.7-fold), Na-K-Cl
cotransporter (NKCC)2 (Slc12a1; 1.09-fold), or Na-Cl cotrans-
porter (NCC) (Slc12a3; 1.24-fold) despite the fact that they are
regulated by corticosteroids and show altered physiological
activity in ACTH-treated mice (6). Nevertheless, the correla-
tion between transcriptional and physiological changes in the
present model has been powerful and has identified important
responses to ACTH in several different areas.
Sodium and water homeostasis. We previously found (6)
that ACTH excess stimulates renal tubular sodium reabsorption
and increases ENaC activity via MR and GR pathways. In the
present study, however, ACTH excess prompted a rapid and
sustained natriuresis, as has been shown in rats (24). There was
no evidence for sodium retention, and glomerular hyperfiltra-
tion may be sufficient to offset enhanced ENaC activity.
Moreover, inhibition of sodium reabsorption in the proximal
tubule is suggested by phosphaturia as well as the decreased
expression of Na/H exchanger (NHE)8 and several organic
anion transporters. Calciuria is consistent with impaired so-
dium reabsorption in the thick limb of Henle, and, combined,
these events may explain the negative sodium balance ob-
served in ACTH-treated mice. The suggestion that corticoste-
roid-induced effects in one nephron region may be compen-
sated by secondary effects elsewhere is illustrated by a recent
study in which human GR was overexpressed in the collecting
duct of mice (33): GR-stimulated gene expression was offset
by opposing changes in upstream nephron segments that did
not express human GR, and blood pressure homeostasis was
preserved. Countervailing effects such as these underpin the
“escape” phenomenon and mitigate the pathophysiological
consequences of long-term corticosteroid excess. Escape from
the sodium-retaining effects of aldosterone is well docu-
mented, and the present study suggests that the kidney also
adapts to excess glucocorticoid. At the molecular level, expres-
sion changes to three genes may combine to limit renal glu-
cocorticoid signaling. First, GR (Nr3c1) was reduced to a level
that, in the hippocampus for example, is sufficient to affect
HPA activity (38). Second, an increase in Fkbp5, which binds
GR, would reduce receptor affinity for glucocorticoids (47). In
squirrel monkeys, for example, Fkbp5 is partly responsible for
glucocorticoid resistance (13). Third, a reduction of Hsd11b1,
which catalyzes the conversion of inactive 11-dehydro deriv-
atives of glucocorticoid to the active parent compound, would
reduce glucocorticoid activity at a local level, particularly in
the renal medulla (10). However, it has been reported that
HSD11B1 functions as a dehydrogenase in human proximal
tubular cells (19), indicating that glucocorticoid activity in
different sections of the tubule may be influenced by local
redox potentials controlling the directionality of conversions
164 RENAL EFFECTS OF CHRONIC ACTH TREATMENT IN MICE
between corticosterone and 11-dehydrocorticosterone as well
as by differences in enzyme expression levels.
In contrast to sodium, ACTH-treated mice had a positive water
balance. This may be consistent with overactivation of MR, but
the effects of GR seem to predominate in terms of plasma volume.
Increased hematocrit is strongly suggestive of a GR influence on
vascular permeability (22), and we recently confirmed (6) con-
traction of plasma volume during ACTH excess. The expression
of genes involved in renal water conservation (Aqp2, Avpr2, and
Slc14a2, for example) was largely unchanged by chronic ACTH
treatment. Aquaporin 4, which is constitutively expressed in the
basolateral membrane of the collecting duct (43), was signifi-
cantly upregulated and may play a permissive role for enhanced
renal water reabsorption.
The complexity of the pathophysiological response to
ACTH excess is illustrated by the separation of sodium and
water homeostasis, and yet the pivotal role of sodium in the
hypertensive response is clear: ACTH did not increase blood
pressure in mice maintained on a diet that was essentially
sodium free (6). We now propose that the development of
hypernatremia is an important hypertensive mechanism: the
redistribution of water outside the vascular compartment
causes contraction-hypernatremia, enhancing the release of
several vasoactive agents (6, 29) and exerting a significant
pressor effect.
Potassium homeostasis. Volume contraction may mean that
potassium depletion is actually more severe than indicated by
plasma concentration alone. Potassium depletion is considered
a hallmark of mineralocorticoid excess, attributable to activa-
tion of MR in the distal nephron, but our data do not support
this hypothesis: ACTH-treated mice indeed maintained an
inappropriately robust potassium excretion, but there was no
kaliuresis and mice were in neutral potassium balance through-
out the experiment. We hypothesize that hypokalemia reflects
a redistribution of potassium out of the vascular space coupled
to a renal conservation defect. The mechanisms underpinning
the redistribution remain unknown, but the renal defect may
reflect both impaired reabsorption through the paracellular
cation shunt in the thick limb of Henle (5) and persistent
secretion in the distal nephron (6). Renal potassium channels
were not identified as differentially expressed by microarray
analysis. The expression level was, however, generally low.
Renal outer medullary potassium channel (ROMK) (Kcnj1;
1.22-fold) was abundantly expressed, but its function is deter-
mined by splice variants and cannot be determined with whole
kidney preparations.
Calcium homeostasis. Microarray analysis revealed novel
effects of ACTH on at least six genes related to calcium
metabolism. Glucocorticoids are well known to inhibit bone
calcium reabsorption, and in the present study the urinary
excretion of calcium and phosphate were both markedly in-
creased by ACTH. Normally, filtered calcium is reabsorbed
along the length of the tubule (30). In the proximal tubule and
thick limb of Henle, which account for the bulk of reabsorp-
tion, calcium is reclaimed by both transcellular and paracellu-
lar mechanisms (15). In the distal convoluted tubule, reabsorp-
tion is exclusively transcellular, involving coordinated regula-
tion of an apical channel, Trpv5, cytoplasmic calbindin 28K
(Calb1), and basolateral Na/Ca exchanger (Slc8a1). In the
distal tubule, calcium reabsorption is regulated by Sgk1: null
mice have reduced levels of Calb1 and Trpv5 (40). In the
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present study, ACTH increased expression of both Sgk1 and
Calb1, which would be predicted to reduce calcium excretion.
However, Trpv5 was downregulated, and Slc8al was not sig-
nificantly affected by ACTH treatment, which may contribute
to calciuria. Regulation by Sgk1 is dependent on the sodium/
hydrogen exchanger regulator Slc9a3r2, which was not stim-
ulated by ACTH treatment, suggesting misregulation of cal-
cium reabsorption in the distal nephron. Indeed, renal calcium
reabsorption is controlled by calcium-sensing receptors within
the kidney and by hormones such as parathyroid hormone and
vitamin D and calcitonin. The microarray data did not reveal
any effect on parathyroid hormone signaling but found in-
creases in three genes associated with vitamin D (Cyp27b1,
Cyp24a1, and Gc). Cyp27b1 encodes the 1␣-hydroxylase en-
zyme that converts 25-hydroxyvitamin D (the main circulating
form of vitamin D) to the biologically active 1,25-dihydroxyvi-
tamin D. Cyp24a1 encodes an inactivating enzyme that hy-
droxylates vitamin D at the 24 position. Gc encodes a high-
affinity serum vitamin D binding protein (DBP). The func-
tional relevance of ACTH-induced changes in these three
genes is difficult to explain. One possibility is that calciuria is
secondary to the glucocorticoid-mediated inhibition of bone
reabsorption. Increased Cyp27b1 could therefore be a compen-
satory mechanism, increasing active vitamin D and preserving
calcium. Although DBP reduces the free active component of
vitamin D in the circulation, it also facilitates the renal pres-
ervation of vitamin D stores and hence will promote the
conservation of calcium (44). DBP also facilitates the uptake of
active vitamin D in target tissues (12). An increase in Cyp24a1,
in contrast, does not fit the hypothesis of compensatory vitamin
D-dependent calcium uptake since 24-hydroxylation is an ef-
fective mechanism of negative-feedback control of 1,25-dihy-
droxyvitamin D synthesis. However, it has been suggested that
Cyp24a1 expression in vitamin D target tissues may have a role
in regulating hormone activity (28).
Xenobiotic metabolism. We anticipated that ACTH would
regulate genes involved in either the production of or the
protection from damaging chemicals: glucocorticoid treatment
is known to increase the synthesis of reactive oxygen species
(21, 36) as well as offering protection in the kidney from
inflammatory cell infiltration following treatment designed to
cause renal tubular damage (35). In the present study, changes
associated with damage limitation predominate, with a group
of 10 or more genes encoding transporters and enzymes in-
volved in xenobiotic metabolism. Interestingly, similar pat-
terns of gene changes have been reported for the genetically
obese and diabetic db/db mouse (11, 35, 48). This suggests
coordinate control possibly mediated by one or more of a group
of established transcription factors (Ahr, Car, Nr1i2, Ppar␣,
Nrf2) for xenobiotic transport genes. However, the expression
of none of these factors was affected in the microarray. The
precise functions of the ACTH-affected xenobiotic transport
genes are not well defined. It seems probable that the trans-
porters are involved in the proximal transport of organic anions
or cations, the clearance of which would be compromised by
glucocorticoid-induced changes in renal function. These en-
zymes may be required to detoxify oxidized lipids and other
active compounds generated as a consequence of glucocorti-
coid effects on metabolism.
Summary. In conclusion, chronic ACTH treatment causes a
sustained increase in circulating glucocorticoids. The pattern of
 RENAL EFFECTS OF CHRONIC ACTH TREATMENT IN MICE
electrolyte excretion and microarray analysis of gene changes
reveal little that could be ascribed directly to MR. Although
GR-dependent changes on renal electrolyte metabolism and
gene transcription were observed, the link between these
changes and blood pressure control will require further inves-
tigation. Many of the gene changes could be considered to be
part of a glucocorticoid escape mechanism. GR signaling
appeared to be downregulated, and at least 10 genes involved
in the metabolism of xenobiotics were affected. The downregu-
lation of Hsd11b1 suggests that local glucocorticoid signaling
may well be altered, and the link between enzyme activity and
altered redox state warrants further investigation. ACTH in-
duced a marked calciuria and phosphaturia. Although genes
controlling vitamin D activity and also renal epithelial calcium
transport were affected, further studies to localize sites of
expression of these genes are needed to determine their role in
causing or counteracting the calciuretic effect of glucocorti-
coids.
ACKNOWLEDGMENTS
We are grateful for the technical assistance of Nicola Wrobel and Carolynn
Cairns and for the microarray work of ARK Genomics at the Roslin Institute.
We thank Forbes Howie for measurement of creatinine and albumin.
GRANTS
D. R. Dunbar was funded by the Wellcome Trust Cardiovascular Research
Initiative and Functional Genomics Initiative. J. J. Mullins is a recipient of a
Wellcome Trust Principal Research Fellowship. L. C. Evans was funded by a
British Heart Foundation Ph.D. studentship. E. A. S. Al-Dujaili, L. J. Mullins,
and C. J. Kenyon were funded by the UK Medical Research Council. M. A.
Bailey was a Wellcome Trust Intermediate Fellow. This work was partially
funded by a British Heart Foundation CoRE Award.
DISCLOSURES
No conflicts of interest are declared by the author(s).
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