Note: Descriptions are shown in the official language in which they were submitted.
DEMANDES OU BREVETS VOLUMINEUX
LA PRESENTE PARTIE I)E CETTE DEMANDE OU CE BREVETS
COMPREND PLUS D'UN TOME.
CECI EST LE TOME DE _2
NOTE: Pour les tomes additionels, veillez contacter le Bureau Canadien des
Brevets.
JUMBO APPLICATIONS / PATENTS
THIS SECTION OF THE APPLICATION / PATENT CONTAINS MORE
THAN ONE VOLUME.
THIS IS VOLUME 1 OF 2
NOTE: For additional volumes please contact the Canadian Patent Office.
CA 02605036 2007-10-01
RENOPROTECTION BY GROWTH HORMONE-RELEASING HORMONE AND
AGONISTS
BACKGROUND OF THE INVENTION
The pituitary growth hormone-releasing hormone receptor (GHRH-R) has been
cloned in several mammalian species, 1-4 including normal human pituitary
2,5.6 and
adenomas. 5"' More recently, GHRH-R was reported in avian 8 and fish
pituitary. 9
The rat pituitary contains a major GHRH-R mRNA transcript (2.5 kb) and a less
abundant one (4 kb; ~20% of the 2.5-kb in 2-month-old rats). 2, 10 While the
2.5-kb
transcript generates the 423 amino acid functional GHRH-R, " the role and
structure
of the 4-kb transcript remain to be elucidated. The 47-kDa-encoded rat protein
belongs to the subfamily B-III of G protein-coupled receptors, which also
include
receptors for VIP, secretin, glucagon, GIP, PTH, calcitonin, CRF and PACAP. 2
In
somatotrophs, the specific binding of hypothalamic GHRH to functional plasma
membrane receptor represents the primary event leading to GH secretion 12-13
and
synthesis 12 mainly through an adenylate cyclase/cAMP/protein kinase (PK) A
pathway 14-1' and possibly a PKC pathway. 18 GHRH-mediated GHRH-R activation
is
also involved in somatotroph proliferation and differentiation via PKA 2, 19-
22 and
mitogen-activated protein (MAP) kinase pathways. 23-24
Apart from the anterior pituitary, a GHRH-GHRH-R system has been identified in
rat
brain, spleen and thymus, ovary, placenta, testis and renal medulla.
Intrasuprachiasmatic/medial preoptic area administration of GHRH stimulates
dietary
protein intake in free-feeding rats 25 while it promotes sleep in the
intrapreoptic
region. 26 In rat spleen and thymus, a functional GHRH-GH axis was shown to
mediate lymphocyte proliferation through a GHRH-induced GH mechanism. 27 In
human and rat reproductive systems, the presence of GHRH-R mRNA 2 and
immunoreactivity 28 has been reported as well as GHRH-mediated effects on
regulation of sex steroid levels, 29 granulosa cell differentiation, 30
placental growth, 31
and gonadotropin stimulation of testosterone. 32
2
CA 02605036 2007-10-01
A functional GHRH-R has been identified in the rat renal medulla. 33, 34
Boulanger et
al. demonstrated the presence of specific, reversible and saturable binding
for [1251-
Tyr10]hGHRH(1-44)NH2 in this tissue. 34 Moreover, stimulation of semi-purified
Henle's loop (HL) cells with GHRH was shown to mediate GHRH-R internalization
and regulation of its expression. 33 The highest level of renal GHRH-R mRNA
was
localized in HL by ribonuclease protection assay and in situ hybridization. 33
Its
localization in HL and the tissue-selective regulation of pituitary and renal
GHRH-R
mRNA levels and its regulation during development and aging may suggests roles
of
GHRH-R in the renal medulla. 33
SUMMARY OF THE INVENTION
In one aspect, the present invention relates to the use of GHRH-receptor
ligand for
preventing (lowering, inhibiting) the death of kidney cells and/or loss of
kidney cell
function associated with oxidative stress.
In an additional aspect, the present invention relates to the use of GHRH-
receptor
ligand for promoting regeneration of kidney cells and/or function in a mammal
in need
thereof.
In a further aspect, the present invention relates to a method of promoting
regeneration of kidney cells and/or function in a mammal in need thereof which
may
comprise administering a GHRH-receptor ligand to the mammal.
In yet a further aspect, the present invention relates to a method of
preventing
(lowering, inhibiting) the death of kidney cells and/or loss of kidney cell
function
associated with oxidative stress, which may comprise the step of administering
a
GHRH-receptor ligand to the mammal.
A mammal in need may be identified by the detection of markers of oxidative
stress
associated with kidney cell damage. These markers may be measures in vivo or
in
bodily fluid such as in urine, serum and plasma. Indicators of deterioration
of Henle's
loop ascending thin limb cells also include change in urine osmolarity,
volume/time
3
CA 02605036 2007-10-01
urine production and content of urine. As such specific markers alone or in
combination with indicators of general kidney function may be used to identify
the
population of patients for which treatment is sought or desirable.
In accordance with the present invention, the GHRH-receptor ligand may be the
native GHRH (SEQ ID NO.:1) or a biologically active fragment. Exemplary
embodiment of GHRH biologically active fragment may include for example, SEQ
ID
NO.:2 or 3.
Also in accordance with the present invention, the GHRH-receptor ligand may be
a
GHRH agonist. Exemplary embodiment of GHRH agonist may include for example,
any one of SEQ ID NO.:4 to 9. Specific embodiments of GHRH agonist may include
for example, any one of SEQ ID NO.:4 to 6 wherein Xaa is absent.
In a further exemplary embodiment of the invention, the ligand may be SEQ ID
NO.:10, wherein Xaa2 is D-Ala and wherein the remaining amino acid sequence is
identical to SEQ ID NO.: 1 or 3.
In an additional exemplary embodiment of the invention, the ligand may be SEQ
ID
NO.: 10, wherein Xaa10 is D-Tyr and wherein the remaining amino acid sequence
is
identical to SEQ ID NO.:1 or 3.
In another exemplary embodiment of the invention, the ligand may be SEQ ID
NO.:
10, wherein Xaa15 is D-AIa and wherein the remaining amino acid sequence is
identical to SEQ ID NO.:1 or 3.
In another exemplary embodiment of the invention, the ligand may be SEQ ID
NO.:10, wherein Xaa22 is Lys and wherein the remaining amino acid sequence is
identical to SEQ ID NO.: 1 or 3.
In yet another exemplary embodiment of the invention, the ligand may be SEQ ID
NO.:10, wherein Xaa2 is D-Ala and/or XaalO and/or D-Tyr and/or Xaa15 is D-AIa
and/or Xaa22 is Lys and wherein the remaining amino acid sequence is identical
to
SEQ ID NO.:1 or 3.
4
CA 02605036 2007-10-01
In an additional embodiment, the ligand may be SEQ ID NO.:10, wherein Xaa8 is
Ala
and/or Xaa9 is Ala, and/or Xaa15 is Ala and/or Xaa22 is Ala.
In yet an additional embodiment, the ligand may be SEQ ID NO.:10, wherein
Xaa22
is Lys.
A mammal at need may be identified, prior to administration of the ligand, by
determining kidney function. More particularly, the mammal may be identified
by
determining the presence of markers associated with oxidative stress to kidney
cells.
The mammal may suffer or may be susceptible of suffering from a disease
selected
from the group consisting aging- and frailty-related nephropathy and renal
failure
nephropathy, diabetes insipidus, diabetes type I, diabetes II, renal disease
glomerulonephritis, bacterial or viral glomerulonephritis, IgA nephropathy,
Henoch-
Schonlein Purpura, membranoproliferative glomerulonephritis, membranous
nephropathy, Sjogren's syndrome, nephrotic syndrome minimal change disease,
focal glomerulosclerosis and related disorders, acute renal failure, acute
tubulointerstitial nephritis, pyelonephritis, GU tract inflammatory disease,
Pre-
clampsia, renal graft rejection, leprosy, reflux nephropathy, nephrolithiasis,
genetic
renal disease, medullary cystic, medullar sponge, polycystic kidney disease,
autosomal dominant polycystic kidney disease, autosomal recessive polycystic
kidney disease, tuberous sclerosis, von Hippel-Lindau disease, familial thin-
glomerular basement membrane disease, collagen III glomerulopathy, fibronectin
glomerulopathy, Alport's syndrome, Fabry's disease, Nail-Patella Syndrome,
congenital urologic anomalies, monoclonal gammopathies, multiple myeloma,
amyloidosis and related disorders, febrile illness, familial Mediterranean
fever, HIV
infection, AIDS, inflammatory disease, systemic vasculitides, polyarteritis
nodosa,
Wegener's granulomatosis, polyarteritis, necrotizing and crescentic
glomerulonephritis, polymyositis-dermatomyositis, pancreatitis, rheumatoid
arthritis,
systemic lupus erythematosus, gout, blood disorders, sickle cell disease,
thrombotic
thrombocytopenia purpura, hemolytic-uremic syndrome, acute cortical necrosis,
renal
thromboembolism, trauma and surgery, extensive injury, burns, abdominal and
vascular surgery, induction of anesthesia, side effect of drug abuse or use of
drugs
CA 02605036 2007-10-01
including those generating renal oxidative stress and toxicity such as
antibiotics and
cancer chemotherapeutic agents, malignant disease, adenocarcinoma, melanoma,
lymphoreticular, multiple myeloma, circulatory disease myocardial infarction,
cardiac
failure, peripheral vascular disease, hypertension, coronary heart disease,
non-
atherosclerotic cardiovascular disease, atherosclerotic cardiovascular
disease, skin
disease, psoriasis, systemic sclerosis, respiratory disease, COPD, obstructive
sleep
apnea, hypoxia at high altitude or endocrine disease,
Examples of markers of kidney function -
Several markers of kidney function are known in the art and markers of
oxidative
stress (damage to DNA, lipids and/or proteins) to kidney cells have been
identified.
As such, urinary measurements of these markers may be useful to identify
patients
for which the present invention is desirable.
In an exemplary embodiment, the total antioxidant status (TAS) of the mammal
may
be measured. This assay is based on the capacity of plasma sample obtained
from
the mammal to inhibit the formation of 2,2'-azinobis (3-ethylbenzothiazoline-6-
sulfonate) (ABTS) radicals in the presence of H202 and metmyoglobine 60. The
percentage of inhibition correspond to the TAS value expressed in Trolox
equivalent.
Upon determining the TAS other plasmatic components are taken into account,
namely; concentration of plasma albumin and uric acid. The TAS will thus be
determined by the following formula:
TAS = TAS measured - f(Albumin mmol/I x 0,69) + Uric acid mmol/I x 1)1
The oxidative capacity of albumin is 0.69 mmol/L Trolox equivalent while the
oxidative capacity of uric acid is lmmol/L Trolox equivalent. Plasma uric acid
levels
may be measured by HPLC.
Oxidative stress to lipids may be determined by evaluating the amount of F2-
isoprostane ( isomers of prostaglandin F2 (PG F2)) which are formed by the non-
enzymatic oxidation of arachnidoic acid under condition of oxidative stress.
More
particularly, 8-iso-PGF2, the most abundant member of this family is a
reliable
marker of in vivo oxidative stress to plasma and cellular lipids. To that
effect, 8-iso-
6
CA 02605036 2007-10-01
PGF2 may be extracted from the organic phase of an esterified urine sample
(with
ester pentafluorbenzyl) and analyzed by gaz chromatography coupled to mass
spectroscopy (GC/MS) as per Nourooz-Zadeh et al.
Oxidative stress to DNA may be determined by measuring the presence of 8-oxo-
dGuo in urine. The presence of this product may be detected by HPLC with
electrochemical detection as per Arthur et al and Reznick et al.
According to the present invention, the term "marker" means any marker of
kidney
function and/or any stress marker known in the art or as described herein.
Stress
markers may be oxidatively damaged proteins and/or lipids, active oxygen
species
(hydroxy radicals, alkoxy radicals, hydroperoxy radicals, peroxy radicals,
iron-oxygen
complexes, superoxides, hydrogen peroxide, hydroperoxides, singlet oxygen, and
ozone) or free radicals (lipid radicals and the like). For example,
concentrations of
two major aldehydic lipid peroxidation (LPO) products, 4-hydroxynonenal (HNE)
and
malondialdehyde (MDA), and of protein carbonyls may be analyzed as parameters
of
oxidative stress related to kidney function. Kidney function markers include
for
example, creatinin, urea, apolipoprotein A-IV. Measurements of these markers
(serum measurement, urinary measurement, etc) may be useful to identify
patients
for which the present invention is desirable.
A high level of GHRH-R mRNA has previously been detected in HL but not in
collecting duct of the rat renal medulla. 33 A functional GHRH-R was also
described in
semi-purified HL cells. GHRH-R immunoreactivity in human whole kidney
preparations 40 and GHRH binding sites in rat medullary homogenates. 34
The present study increases our knowledge on the medullary GHRH-R, in
identifying
its cell-specific localization in HL. Using preparations of purified thin and
thick limbs of
Henle's loop cells, a high level of GHRH-R mRNA was detected in thin limbs
only.
Since thin limb cells contains a descending segment participating to water
transport,
and an ascending segment actively involved in ion transport, it was important
to
identify the specific cell type expressing GHRH-R in this part of the nephron,
to help
defining potential roles. Co-immunolocalization of GHRH-R, with specific
markers of
7
CA 02605036 2007-10-01
descending (aquaporin-1) 41 and ascending (CIC-K1) 36, 42 thin limb cells was
performed. GHRH-R immunofluorescence was highly co-localized with that of CIC-
K
but not aquaporin-1, indicating a specific expression in ascending thin limb
cells.
Moreover, our results show, for the first time, the presence of a local GHRH-
GHRH-R
system in these cells.
To gain more information on potential roles of the renal GHRH-R, the
regulation of
medullary GHRH-R mRNA levels was studied using in vivo models of Na+/CI" or
water homeostasis disruption, as obtained with a high-NaCI diet for 2, 7 or 14
days or
a water deprivation for 3 or 5 days. At the cellular level (GHRH-R mRNA level
per
fixed amount of total RNA), GHRH-R mRNA concentrations were differentially
regulated according to the duration of the high-salt diet. They decreased at 2
days,
increased at 7 days, and returned to normal at 14 days. The same type of
change
was seen when data were analyzed at the tissue level (GHRH-R mRNA levels per
total medulla RNA content), indicating that the cellular effect was not
counterbalanced systemically. This regulation of GHRH-R mRNA levels was
reflected
in the sensitivity of GHRH to induce cAMP production in freshly dispersed thin
limb
cells from rats submitted to the high-NaCl diet, in comparison to those fed
the control
diet. After 2 days of high-salt diet, a stimulation with either a low (1 nM)
or high (100
nM) concentration of GHRH resulted in a decreased production of cAMP,
correlating
with that of GHRH-R mRNA levels. After 7 days, GHRH-induced cAMP levels were
restored, indicating that an increased production of GHRH-R mRNA may be
necessary to rapidly restore GHRH sensitivity and likely GHRH-R functional
receptor
levels. Up to now, no data has been reported on the effect of high-sait-
induced
oxidative stress in thin limb cells, specifically.
The effects of the high-NaCI diet were not mimicked by a 3- or 5-day water
deprivation, two situations provoking hypertonicity. Therefore, it indicates
that the
ascending thin limb GHRH/GHRH-R system is not directly involved in the
regulation
of ion transport. The GHRH-R could rather participate to adaptive processes in
ascending thin limb cells to compensate for an increased oxidative stress and
cell
damage caused by a drastic and sustained high-NaCI intake. 45 High-salt diet
regulates genes involved in higher fibrotic activity, cellular stress and
apoptotis in the
rat renal medulla. 46 and administration of substances exhibiting antioxidant
properties attenuates or prevents these deleterious effects. 47,48 Changes in
GHRH-R
8
CA 02605036 2007-10-01
mRNA levels and GHRH sensitivity, between 2 and 7 days of a high-NaCl diet,
suggests that GHRH-R activation may promote ascending thin limb cell survival
early
on in a situation of oxidative stress and subsequently proliferation. A sc
administration of GHRH, once a day from the beginning of a 2-day high-NaCI
diet,
increased markedly the number of ascending thin cell nuclei and mitochondria
immunolabeled to BrdU. In addition, the intensity of anti-BrdU labeling was
significantly augmented in the cytoplasm co-labeling with MitoTracker red
CMXRos, a
reliable indicator of functional mitochondria. Thus, in condition of oxidative
stress,
activation of the renal GHRH-R plays a role in adaptive processes related to
DNA
repair and/or synthesis, leading to cell survival and subsequent proliferation
of these
squamous epithelial cells not very rich in mitochondria. Stimulation with
exogenous
GHRH therefore accelerates or potentiates these processes, with up-regulation
of the
GHRH-R and CIC-K1, essential in ascending thin limb functions. Indeed, we have
demonstrated that GHRH directly induces thin limb cell proliferation in vitro.
No
significant regulatory effect was seen on anterior pituitary GHRH-R mRNA
levels with
a 2-day in vivo of GHRH (data not shown). We have previously shown that IGF-I
serum levels are significantly decreased after a 14-day sc administration of 1
mg/kg
BW/day rGHRH(1-29)NH2 but not with 0.5 mg /kg BW/day). 10 Therefore, the
dosage
and duration used to regulate the renal GHRH-R will not regulate the pituitary
GHRH-
R.
Oxidative stress occurs inside cells or tissues when production of oxygen
radicals
exceeds their antioxidant capacity. Excess of free radicals damage essential
macromolecules such as protein, lipids and DNA, leading to abnormal gene
expression, disturbance in receptor activity and signaling, apoptosis,
immunity
perturbation, mutagenesis, and protein or lipofushin deposition. Numerous
human
diseases involve localized or general oxidative stress. In many serious
diseases such
as cancer, ocular degeneration (age-related macular degeneration or cataract)
and
neurodegenerative diseases (ataxia, amyotrophic lateral sclerosis, Alzheimer's
disease), oxidative stress is one of the primary factor. In various other
diseases,
oxidative stress occurs secondary to the initial disease and plays an
important in role
in immune and vascular complications, such as in AIDS, septic shock,
Parkinson's
disease, diabetes and renal failure. 49, 50 It is also the case in aging, were
9
CA 02605036 2007-10-01
accumulation of cellular oxidative stress is considered as a key element in
the
deterioration of tissues, organs and systems. 51
In the present study, the pituitary GHRH-R, which is exclusively localized on
somatotroph cells, 52 was found to be insensitive to the high-salt diet,
contrarily to
that of ascending thin limbs, demonstrating the vulnerability of the latter. A
tissue-
specific regulation of renal medulla and anterior pituitary GHRH-R mRNA levels
has
previously been shown in developing and aging rat. 33 Whether or not
somatotroph
sensitivity to GHRH can be altered during the first 2 days of the high-NaCI
diet,
without affecting GHRH-R mRNA levels, remains possible. In contrast, a water
deprivation strongly increased pituitary cell and tissue GHRH-R mRNA levels.
Since
it induces a drastic reduction of food intake and that dietary protein
restriction down-
regulates hypothalamic preproGHRH mRNA, 53 a subsequent decrease of pituitary
GHRH-R may have occurred. These results suggest that somatotroph and
ascending thin limb cell GHRH-R mRNA levels may be primarily regulated by
hypothalamic and renal GHRH, respectively. Difference in the primary structure
of
the pituitary and renal GHRH-R and/or the relative abundance of the native 423-
aa
GHRH-R and isoforms may also contribute to a tissue-specific regulation. In
the rat
pituitary, apart from the 423-aa GHRH-R, two splice variants have been
identified z,
11, 54 but their relative abundance has not been quantified rigorously. The
464-aa
variant bears a 41-aa addition inserted into the 3rd IC domain, 11 while the
480-aa
variant bears the long 3rd IC loop and a modified C-terminus, resulting from a
131-bp
deletion (nt 1279-1408). 54 GHRH binds with moderate affinity to the 464-aa
variant,
transiently transfected in HeLa cells, and induces 55 or not 11 cAMP
production. The
ability of GHRH to stimulate cAMP was reported to be lower with the 480-aa
variant
than the 464-aa variant, 54 suggesting that the 3rd IC loop and the C-terminus
are
critical for GHRH-activation of the cAMP-AC-PKA pathway.
Finally, this action of GHRH is mediated in rat ascending thin limb cells by a
GHRH-R
exhibiting a 5' DNA sequence different from that of the rat anterior pituitary
GHRH-R.
This structural difference in the rat renal GHRH-R compared to the pituitary
GHRH-R
was also observed for the murine (data not shown) and porcine renal GHRH-R. As
porcine and human pituitary GHRH-R share the highest sequence identity (86%),
2, 3
CA 02605036 2007-10-01
it is suggested that the renal GHRH-R variant found in the rat medulla is also
present
in human renal medulla.
BRIEF DESCRIPTION OF THE FIGURES
Fig. 1. GHRH-R mRNA levels in the renal medulla and purified thin and thick HL
cells form 2-month-old healthy rats. Five (thin HL) and 20 g (thick HL and
medulla)
of total RNA were analyzed by RPA. Results were expressed as per 20 g of
total
RNA, in percentage of relative density to that obtained in 20 g total RNA
samples
from the medulla. Results represent the mean SEM of samples analyzed in
duplicate from 2 independent RPA experiments and were normalized with both
GAPDH and the cRNA external standard.
**P <0.01 when compared to GHRH-R mRNA levels in total medulla (Dunnett's
test).
Fig. 2. Immunocytochemical localization of the GHRH-R in purified thin limb
cells
form 2-month-old healthy rats. Co-localization of GHRH-R immunofluorescence
(b, e)
was assessed in renal cells from 2-month-old male rats using an anti-aquaporin-
1
antibody, as a marker of descending thin limb cells (a) and an anti-CIC-K
antibody,
as a marker of ascending thin limb cells (d). Immunolabeling was specific and
not
labeling was observed when substituting the anti-GHRH-R(392-404) Ab for normal
IgGs (data not shown). Nuclei were labeled with DAPI. Results are
representative of
three independent experiments.
Fig. 3. Visualization of immunoreactive GHRH and CIC-K1 chloride channel and
PCR amplification of preproGHRH in purified thin limb cells from 2 month-old
healthy
rats. A) Labeling of GHRH (a, b) and the CIC-K1 chloride channel (c, d) was
performed in purified thin limb cells using an anti-rat GHRH(1-43)OH Ab and an
anti-
CIC-K Ab. Overlay of GHRH and CIC-K immunofluorescence is shown in f. The
specificity of labeling was assessed by substituting the anti-rat GHRH(1-43)OH
Ab for
normal IgGs (e). B) Representative agarose gel electrophoresis of preproGHRH
and
GAPDH PCR products and molecular weight markers. GAPDH sense 5'-
gggtgtgaaccacgagaaat-3', GAPDH antisense 5'-actgtggtcatgagcccttc-3', nt 1242-
1376 (NM_017008); preproGHRH sense 5'-atgccactctgggtgttcttt-3', preproGHRH
antisense 5'-gcagtttgcgggcatataat-3', nt 196-352 (NM_031577).
11
CA 02605036 2007-10-01
Fig. 4. Effect of a 2-, 7- or 14-day 8%-NaCI dietary intake on medullary GHRH-
R
mRNA levels from 2-month-old rats. A) Autoradiographic representation of GHRH-
R
mRNA, GAPDH mRNA and RPR-64 Msc I cRNA external standard (40 pg) signals
analyzed by RPA, from rats fed 8%- or 0.3%-NaCI (control) diet. B) GHRH-R mRNA
levels expressed per 20 g total RNA. For the 2-day experiment, 5-6 individual
rats
were used in each group for both RPA and statistical analysis, while for the 7-
and
14-day experiment, 7-8 individual rats were used. Results are expressed in
percentage of relative density to that obtained in the medulla from control
rats and
represent the mean SEM of individual samples from each group, analyzed in
triplicate twice and normalized with GAPDH and the cRNA external standard.
*P <0.05 and **P <0.01 when compared to GHRH-R mRNA levels in the medulla
from control rats (Student's t test).
Fig. 5. Effects of a 2-, 7- or 14-day 8%-NaCI dietary intake and a 3-day water
deprivation on anterior pituitary GHRH-R mRNA levels from 2-month-old rats. A-
D)
GHRH-R mRNA levels analyzed by Northern blotting and expressed per 12 g total
RNA. For the 2- (A), 7- (B) and 14-day (C) 8%-NaCI experiment, 7-8 individual
rats
were used in each group for both Northern blotting and statistical analysis,
while for
the 3-day water deprivation (D), 3 (controls) and 7 (deprived) individual rats
were
used. Results are expressed in percentage of relative density to that obtained
in the
pituitary from control rats and represent the mean SEM of individual samples
from
each group, analyzed in duplicate and normalized with normalized with rRNA
28S.
*P <0.05 and ***P <0.001 when compared to GHRH-R mRNA levels in the pituitary
from control rats (Student's t test).
Fig. 6. Basal and GHRH-stimulated cAMP levels in semi-purified thin limb cells
from 2-month-old rats, following a 2-, 7- or 14-day 8%-NaCI dietary intake.
Basal and
net GHRH-stimulated cAMP levels were quantified by EIA (fmol/ g prot) in
freshly
dispersed semi-purified thin limb cells of rats fed 2- (A), 7- (B) or 14- (C)
days a 8%-
or 0.3%-NaCI (control) diet. Results are expressed in percentage of control
values
both for basal and stimulated cAMP levels. Cells from 4 individual rats were
used in
each diet and control group.
12
CA 02605036 2007-10-01
*P <0.05 and **P <0.01 when compared to cAMP levels in semi-purified thin limb
cells from control rats (Student's t test).
Fig. 7. Effect of a GHRH in vivo sc administration of GHRH in 2 month-old rats
fed
a 8%- or 0.3%-NaCI diet on anti-BrdU labeling. BrdU was injected ip 2 h prior
sacrifice (100 mg/hg BW). Rats were fed a 8%- or 0.3%-NaCI (control) diet and
concurrently injected with rGHRH(1-29)NH2 (1 mg/kg BW). Purified thin limb
cells
were cultured 16 h on coverslips and processed for limmunocytochemistry. A)
Increased number of cells exhibiting specific anti-BrdU labeling, colocalizing
either
with DAPI (nuclear) or Mitotracker red CMXRos (mitochondrial) and B) increased
anti-BrdU total fluorescence intensity in nuclear or mitochondrial compartment
were
expressed in percentage of control values (0.3%-NaCL salt diet, GHRH vehicle
injection).
*P <0.05 when compared to levels in purified thin limb cells from control rats
(Dunnett's t test).
Fig. 8. Effect of a GHRH in vivo sc administration in 2 month-old rats on the
regulation of GHRH-R and CIC-K1 mRNA levels in purified thin limb cells. Two-
month-old healthy male Sprague Dawley rats, received a subcutaneous
administration of rGHRH(1-29)NH2 (1 mg/kg BW/day) or the saline vehicle for 2
days.
(A) GHRH-R and CIC-K1 (B) mRNA levels were analyzed by real-time RT-PCR.
Eight animals were used in control and treatment. Group 1 = 3 rats. Group 2 =
5 rats.
*P <0.05 and **P <0.01 when compared to levels in purified thin limb cells
from
control rats (Dunnett's t test).
Fig. 9. RT-PCR products from rat and porcine renal medulla and anterior
pituitary
obtained with a panel of primers of the pituitary GHRH-R. A) Rat and B)
porcine total
RNA was used. Lanes 1, 2: sense and antisens 5' end primers, lanes 3, 4: sense
and
antisens middle portion and lanes 5, 6: sense and antisens 3' end primers,
respectively.
Fig. 10. rGHRH(1-29)NH2-induced cell proliferation in semi-purified thin limb
cells.
Proliferation was assessed after a 60-h cell culture period, using a CeIlTiter
96R
Aqueous one solution cell proliferation assay. Results represent the mean
SEM of
13
CA 02605036 2007-10-01
2 independent experiments performed in duplicate. *P< 0.05, **P< 0.01 when
compared to control levels (Dunnett's t test).
DESCRIPTION OF THE INVENTION
This invention will be described herein below, by reference to specific
examples,
embodiments and figures, the purpose of which is to illustrate the invention
rather
than to limit its scope.
MATERIALS AND METHODS
Animal handling, treatments and tissue preparations
Two-month-old male Sprague Dawley rats (Charles River Canada, St-Constant, QC)
were kept in temperature- (22 C), humidity- (65%) and lighting- (12-h cycles;
lights
on at 0700 h) controlled rooms and had free access to standard rat chow (2018
Tecklad global 18% protein rodent diet, containing 0.23% Na+ and 0.4% CI";
Harlan
Tecklad, Madison, WI) and tap water. Rats were acclimatized ~3 days before
going
on a high-NaCI diet or water deprivation. Rats fed the custom made high-NaCI
diet
for 2, 7 or 14 (8% NaCi; Harlan Tecklad) were compared to rats fed the custom
made
control diet (0.3% NaCI; Harlan Tecklad). They had free access to water. Rats
deprived of water for 3 or 5 days had free access to 2018 Tecklad rat chow.
Rats
used in the first series of experiments, to quantify GHRH-R mRNA levels
following a
8%-NaCI diet or a water deprivation, were housed individually in metabolic
cages for
the entire duration of intervention. BW, food and water intakes, and urine
volume
were recorded daily, and Na+ levels were analyzed on the last 24-h urine
sample
before sacrifice. Rats used in the 8%-NaCI diet/GHRH study were housed
individually
in plastic cages and BW and food intakes were recorded daily. Rats were
sacrificed
in a block-design fashion between 0900-1130 h, by rapid decapitation.
Pituitaries,
kidneys and livers were excised immediately and anterior pituitaries and renal
medullas dissected out. Tissues were snap-frozen in liquid nitrogen and stored
at -
80 C until RNA extraction. For isolation of thin and thick limb cells, renal
medullas
were dissected out rapidly, washed and minced in ice-cold oxygenated HEPES-
Ringer buffer (290 mosm, pH 7.4). For isolation of thick ascending limb cells,
inner
stripes of outer medullas were dissected out and kept in ice-cold oxygenated
Hanks
14
CA 02605036 2007-10-01
solution. 55 For in vivo BrdU-Iabeling experiments, rats were fed a 0.3%- or
8%-NaCl
chow for 2 days (day 1, day 2) and received in the back a subcutaneous (sc)
injection
of 1.0 mg rGHRH(1-29)NH2/kg BW, solubilized in normal physiological saline
(GHRH-treated) or an isovolumetric amount of saline (control). rGHRH-(1-29)NH2
(synthesized in our laboratory) 56 was solubilized each morning just before
treatment
and kept on ice. Rats were injected intra-peritoneally on the morning of day
3, with
100 mg 5-bromo2'-deoxy-uridine/kg BW (30 mg ultrapure BrdU/1 ml in normal
saline;
Sigma-Aldrich Canada Ltd, Oakville, ON), 2 h prior to sacrifice. For in vivo
GHRH
treatment, rats received in the back a subcutaneous (sc) injection of 1.0 mg
rGHRH(1-29)NH2/kg BW daily.
Porcine anterior pituitaries and renal medullas from Yorkshire-Landrace pigs
('007
kg, ;0 50-day-old) were dissected out at a local slaughter house, snap-frozen
in liquid
nitrogen and stored at -80 C until RNA extraction.
Isolation of thin limbs of Henle's loop cells
Cell dispersion of minced medullas to obtain semi-purified thin limb cells was
performed as previously described. 33 These cells were used immediately for in
vitro
determination of basal and rGHRH(1-29)NH2-induced cAMP levels or purified by
differential centrifugation for immunocytochemistry, using a continuous
gradient of
Nycodenz. The gradient was prepared as described by Grupp et al. 55 and thin
limb
cells were recovered in fraction I of the gradient after centrifugation at
1500 g(16 C,
45 min) and washed twice in HEPES-Ringer buffer (430 g, 16 C, 10 min). Cell
viability, assessed by the Trypan Blue exclusion method, was around 95%. When
purified thin limb cells were cultured, isolation and purification steps were
performed
under sterile conditions and media containing antibiotics.
Isolation of thick limbs of Henle's loop cells
The inner stripe of outer medullas were dissected out using an optical
stereomicroscope, minced and kept in oxygenated Hanks solution. Short time
cell
dispersion was performed as previously described, 57 and dispersed cells were
poured on the top of a 100 m-pore nylon membrane (Millipore, Nepean, ON, CA)
and washed with Hanks-1% BSA (Sigma-Aldrich) solution using a syringe adapted
to
a 25G needle. Thick ascending HL cells were detached from the membrane by
CA 02605036 2007-10-01
washing with Hanks-1 % BSA solution. The suspension was centrifuged at 80 g
for 5
min (4 C) and the pellet resuspended in ice-cold Hanks solution. Cell
viability was
determined as above and was similar.
Immunocytochemical procedures
Specific markers of descending (anti-aquaporin-1 antiboby (Ab)) and ascending
thin
limb (anti-CIK-K1/-K2 (CIC-K) Ab) cells (Alamone Labs, Jerusalem, Israel) were
directly conjugated to the fluorochrome Alexa 488, using the AlexaTM488
Protein
Labeling Kit (Molecular Probes, Eugene, OR), according to the manufacturer's
protocol. Labeled antibodies were purified on molecular size exclusion spin
columns,
supplied with the kit (1100 g, 5 min). Purified thin limb cells were fixed in
fresh 4%
paraformaldehyde-phosphate-buffered saline (20 min, RT), washed twice with PBS
and centrifuged (800 g, 4 C, 5 min). Thin limb cells (Q00,000) were spun onto
glass
slides by cytocentrifugation (32 g, RT, 2 min) and permeabilized in 0.2%
Triton X-1 00
(Sigma-Aldrich) for 15 min (RT). Slides were washed in PBS (4 x 5 min, RT),
blocked
with 5% (wt/vol.) BSA-PBS (30 min, RT) and washed in PBS (2 x 10 min). GHRH-R
was detected using 0.5 g of the purified anti-GHRH-R(392-404) polyclonal
antibody
33 in 100 l PBS, containing 1% BSA, incubated overnight (ON) at 4 C, in a
humid
atmosphere (7, 8). Cells were rinsed in PBS (2 x 10 min, RT), incubated 60 min
(RT), in the presence of Alexa 568TM-conjugated goat anti-rabbit IgGs
(Molecular
Probes) (1:15000 in PBS-BSA 1% buffer) and washed in PBS (2 x 10 min).
Descending thin limb cells were then visualized using a rabbit polyclonal
Alexa
488TM-conjugated anti-aquaporin-1 antibody (1:2000 diluted in PBS-BSA 1%, 60
min,
37 C) while ascending thin limb cells were visualized using a rabbit
polyclonal Alexa
488TM-conjugated anti-CIC-K antibody (1:500 diluted in PBS-BSA 1%, 60 min, 37
C).
A final wash of slide-mounted cells was done in PBS (2 X 10 min). Specificity
of
labeling was assessed by substituting GHRH-R (392-404) polyclonal antibodies
with
normal IgGs. Another series of experiments, using a similar procedure as
described
above, was performed to determine whether or not immunoreactive GHRH was
present in ascending thin limb cells. An anti-rat GHRH(1-43)OH antibody (0.5
g/ 100
l; Bachem Biosciences Inc, King of Prussia, PA ) and a secondary Alexa 568-
conjugated goat-anti-rabbit IgGs (1/7500, Molecular Probes) were used. One M
of
4,6-diamodino-2-phenylindole dihydrochloride (DAPI, Molecular Probes) was
added
16
CA 02605036 2007-10-01
for the last 30 min of incubation to stain nuclei. All procedures with
fluorescent
probes were performed in the dark. Cells were visualized using a Nikon Eclipse
E600
(Nikon Canada Inc., Montreal, QC) fluorescence/light microscope equipped with
filters for excitation/emission of fluorescein (485/520 nm) and Texas Red
(595/660)
and DAPI (360/460 nm).
BrdU immunolabeling (Roche Diagnostics, Laval, QC, CA) was used to quantify
DNA
repair/synthesis in purified thin limb cells from rat submitted 2 days to a 8%-
or 0.3%-
NaCI diet and injected with GHRH or saline. The cells were purified as above,
cultured 16 h in DMEM/F12, containing 25 mM glucose, 10% fetal bovine serum,
1%
penicillin-streptomycin, 0,1% amphotericin, 33 on coverslip in 24-wells
sterile culture
plates (=1 X 106 cells/well). They were fixed with fresh 4% paraformaldehyde
(500
l/well, 15 min, RT) and washed using the washing buffer supplied with the kit
(2X5
min, 500 l/well). They were subsequently incubated in blocking buffer as
above (30
min, RT, 500 l/well). Immunolabelling was performed with the primary antibody
anti-
BrdU (dilution 1:10 in the incubation buffer, 30 min, 37 C, 150 l/well). Non
specific
fluorescence was determined by substituting the primary antibody by normal
rabbit
IgGs. Immunodetection was performed after washing by adding a secondary anti-
rabbit-IgG antibody coupled to fluorescein (30 min, 37 C, 150 l/well). All
steps using
fluorescent labeling was performed in the dark. Nuclei and mitochondria were
labeled
using 1 M DAPI and 10 nM of Mitotracker red CMXRos (Molecular Probes, Oregon,
USA; PBS 1X, 15 min, RT, 200 l/well). After final washing, cells on
coverslips were
dried and mounted with Prolong mounting medium/Prolong antifade (Molecular
Probes). Slide-mounted cells were kept 16-24h at RT and stored at 4 C in the
dark.
Cells were visualized and fluorescence intensity was quantified using
fluorescence
microscopy as described above. Intensity of fluorescence and occurrence of co-
labeling were analyzed using the Metamorph 4.5 software (Universal Imaging
Corporation, Canberra Packard Canada LTD, Mississauga, ON, CA. A 6-level (0 to
5)
intensity scale was used to assess fluorescence intensities: background (level
0) : 0-
43 pixels, very weak (level 1); 44-85 pixels, weak (level 2); 86-128 pixels,
moderate
(level 3); 129-170 pixels, high (level 4); 171-213 pixels and very high (level
5): 214-
255 pixels), as previously described. 58 Total fluorescence was determined for
each
17
CA 02605036 2007-10-01
image using arbitrary density units defined as : E(% cell labeled X intensity
level).
Levels 3-5 were considered as immunospecific.
Ribonuclease protection assay of renal GHRH-R
Total RNA from medullas and purified descending and ascending thin limb cells
was
extracted with TRIzol (Invitrogen Canada, Burlington, ON). GHRH-R mRNA levels
were assessed using the RPR64 probe corresponding to the 3'-end of the rat
GHRH-
R complementary DNA (cDNA) (nucleotide position: 1044-1611; Genbank accession
number: L01407). 2 The ribonuclease protection assay was performed as
previously
described. 33 Twenty g total RNA from renal medulla or purified thick HL or
liver, 5
g total RNA from purified thin limb cells or anterior pituitary were used.
Tissue
GHRH-R and GAPDH mRNA and cRNA external standard levels were quantified by
densitometry. GHRH-R mRNA levels were always normalized with both GAPDH
mRNA internal and GHRH-R cRNA external standards, in order to maintain an
intra-
assay coefficient of variation <_ 10% in all experiments. Specificity of the
[32P]GHRH-
R probe was assessed in each experiment using positive (5 g pituitary total
RNA)
and negative (20 g liver total RNA) controls. In addition, linearity of
protected signals
was assessed in each experiment, using 10-30 g medulla total RNA. Results
were
expressed in percentage of relative density to the control condition or tissue
preparation, using a fixed amount of total RNA, which reflects the
concentration of
GHRH-R mRNA at cellular level. Since changes may either be compensated or
aggravated at the organ/tissue level, results were also expressed as total
GHRH-R
mRNA relative densities per renal medulla total RNA content, to document
physiological impacts of interventions. 33
Northern blot hybridization of anterior pituitary GHRH-R
Total RNA was extracted as above. Northern blot hybridization was performed as
previously described on 12 g total RNA samples with minor modifications. 33
Prehybridization was performed in Robbins' hybridization solution (7% SDS
containing 0.25 M Na2HPO4 (pH 7.4), 1 mM EDTA (pH 8.0) and 1% BSA) at 65 C, 2
h. Hybridization was performed in fresh Robbins' solution at 65 C (ON), in the
presence of labeled RPR64. Membranes were subsequently washed, exposed to
films, stripped and rehybridized with GAPDH 28S probes. 33 Quantification of
each
18
CA 02605036 2007-10-01
GHRH-R mRNA transcript (2.5 and 4 kb), GAPDH mRNA and 28S rRNA levels was
performed by densitometry. GHRH-R mRNA levels were normalized with 28S rRNA
in all experiments, to maintain the intra-assay coefficient of variation <_
10%.
Specificity of the [32P]RPR64 cDNA probe was assessed in each experiment using
5
g liver total RNA. Linearity of protected signals was measured routinely,
using 6-18
g total RNA. Results were expressed in percentage of relative density to that
of
control groups, using a fixed amount of total RNA. Results were also expressed
as
total GHRH-R mRNA relative densities per anterior pituitary total RNA content.
Reverse transcriptase-PCR of preproGHRH
Total RNA from purified thin limb cells (2 g) was subjected to two steps RT-
PCR
using SuperScriptTM First-Strand Synthesis System (Invitrogen). Reverse
transcription was performed using SuperScriptTM II RT and PCR reaction was
performed using Platinum Taq DNA polymerase according to manufacturer's
protocol (First-Strand synthesis using oligo(dT) PCR for targets up to 4 kb).
PCR
reaction was performed on a 1:5 dilution of the first strand cDNA product in a
final
volume of 50 I containing 0.4 l of Platinum Taq DNA polymerase. Reagents
were
added to a final concentration of 1X PCR buffer [20 mM Tris-HCI (pH 8.4), 50
mM
KCI], 1.5 mM MgCI2, 0.2 mM dNTPs and 0.3 M sense and antisense desalted
primers diluted in sterile picopure water (GAPDH sense 5'-gggtgtgaaccacgagaaat-
3',
GAPDH antisense 5'-actgtggtcatgagcccttc-3', nt 1242-1376 GenBank NM_017008;
preproGHRH sense 5'-atgccactctgggtgttcttt-3', preproGHRH antisense 5'-
gcagtttgcgggcatataat-3', nt 196-352 GenBank NM_031577). The reaction was
performed in Biometra TGradient PCR (Montreal Biotech Inc, Montreal, QC) with
the
following cycle profile: denaturation at 94 C for 2 min, followed by 39 cycles
of
denaturation at 94 C for 30 sec, annealing at 58 C for 70 sec, extension at 72
C for
60 sec and a final cycle at 94 C for 30 sec, 58 C, 60 C, and 62 C for 60 sec
and a
5-min extension at 72 C. PreproGHRH and GAPDH PCR products were analyzed by
gel electrophoresis on 2% agarose gel containing 0.5 g/ml of ethidium bromide
with
a 100 bp molecular weight standard (Invitrogen).
19
CA 02605036 2007-10-01
RT-PCR of GHRH-R
Total RNA (2ug) from purified aTL cells was subjected to two steps RT-PCR
using
SuperScriptTM First-Strand Synthesis System for RT-PCR (Invitrogen/Canada Life
Technologies). Reverse transcription was performed using SuperScriptTM II RT
and
PCR reaction was performed using Platinum Taq DNA polymerase according to
manufacturer's protocol (First-Strand synthesis using oligo(dT) or GSP and PCR
for
targets up to 4 Kb). PCR reaction was performed on a 1:5 dilution of the first
strand
cDNA product in a final volume of 50 uL containing 0.4 ul of Platinum Taq DNA
polymerase. Reagents were added to a final concentration of 1X PCR buffer (20
mM
Tris-HCI (pH 8.4), 50 mM KCI], 1.5 mM MgCI2, 0.2 mM dNTPs) and 0.3 uM sense
and antisense desalted primers from the rat (three sets of primers covering
the 5',
middle portion and 3' regions were used (PubMed NM_012850): nt 58-191 (5'-
ctctgcttgctgaacctgtg-3' (sense (s)), 5'-catcccatggacgagttgtt-3' (antisense
(as)); nt
578-736 (5'-ctgctgtcttccagggtgat-3' (s), 5'-taggagatgtggaggccaac-3'(as)); nt
1064-
1227 (5'-acttcctgcctgacagtgct-3' (s), 5'-tggcagaagttcagggtcat-3' (as)), and
porcine
pituitary GHRH-R (three sets of primers three sets of rat primers covering the
5',
middle portion and 3' regions were used (PubMed L11869: nt 144-271 (5'-
ctgctgagctccctaccagt-3' (s), 5'-cagcccgaggaggagttg-3' (as)); nt 694-816 (5'-
gcttctccacggttctgtgca-3' (s), 5'-tgggtgacgtagaggccaag-3'(as)); nt 1201-1342
(5'-
gctccttccagggcttcattgt-3' (s), 5'-gaaggctttgcccatttggca-3' (as)) cDNA sequence
were
diluted in sterile picopure water. The reaction was performed in Biometra
TGradient
PCR (Montreal Biotech Inc) with the following cycle profile: denaturation at
94 C for 2
min, followed by 39 cycles of denaturation at 94.0 for 30 sec, annealing at
58.0 C,
60.0 C, and 62.0 C for 70 sec, extension at 72 C for 60 sec and a final cycle
at 94 C
for 30 sec, 58.0 C, 60.0 C, and 62.0 C for 60 sec and a 5-min extension at 72
C.
GHRH-R, GAPDH and GHRH PCR products were analyzed by gel electrophoresis
on 2% agarose gel containing 0.5 ug/mI of ethidium bromide with a 100 bp
molecular
weight standard (Invitrogen Life Technologies, Burlington, ONT, CA). The gel
was
visualized using a IS1000 Digital imaging system (Alpha Innotech
Corp./Canberra
Packard).
Quantitative real-time RT-PCR of GHRH-R and CICK-1
CA 02605036 2007-10-01
Total RNA from purified thin limb cells was extracted with TRlzol. Samples
were
resuspended in RNAse-free water (Ambion). Reverse transcription of 2 g total
RNA
was performed using the SuperScriptTM II RT kit (Invitrogen) and random
hexamer
primers, according to the manufacturer's protocol, including RNAse H
treatement.
mRNA levels of rat GHRH-R and rat GAPDH (internal control) were determined in
separate tubes, by real-time PCR, using a 1/150 (GHRH-R) and 1/300 (GAPDH)
dilution of the RT product and the reagents from the QuantitectTM SYBR Green
PCR kit (Qiagen, Mississauga, ON, CA), according to the manufacturer's
recommendation. The ABI protocol was used except that the dUTP/uracil-N-
glycosylase step was omitted. Reactions were performed in duplicate, in a
final
volume of 25 L, containing 300 nM of sense and antisense primers, using a
Rotor
Gene 3000 real-time thermal cycler (Montreal Biotech Inc, Montreal, QC, CA).
No
template and no amplification controls were always included to confirm the
specificity
of reactions. The parameters included a single cycle of 95 C for 15 min,
followed by
45 cycles of 94 C for 15 sec, annealing at 52 C for 30 sec, extension at 72 C
for 30
sec and a melting step going from 72 C to 99 C (ramping at 1 C/sec). Specific
primers (300 nM) for GHRH-R , CICk-1 and GAPDH were used. Specificity of the
PCR products was established by melting curve analysis and by running products
on
2% agarose gel, containing 0.5 g/ml of ethidium bromide, with a 100 bp
molecular
weight standard (Invitrogen). Results were analyzed using the Rotor-Gene
application software (version 6.0). A five-point standard curve was performed
for
each gene tested, using 1:5 serial dilutions (1:5 to 1:3125) of renal medulla
total RNA
from 2-month-old healthy male rats. The intra-assay coefficient of variation
of GHRH-
R and GAPDH Ct values was <_ 2.5% in all experiments
Quantification of cAMP levels in semi-purified thin limbs of Henle's loop
cells
Sensitivity to GHRH was assessed in freshly semi-purified thin limb cells 33
from rats
fed a 8%-NaCI diet for 2, 7 or 14 days or the control diet. Cells (1X106 cells
= 60-75
g prot/ml/Eppendorf tube) were preincubated 30 min (37 C) in 1 ml DMEM/F12
cultured media, 33 containing 1X antibiotics, 0.2%BSA and 1 mM isobutyl-l-
methylxanthine (IBMX, Sigma) and challenged 15 min (37 C) with 1 and 100 nM
GHRH, the vehicule (DMEM/F12-0.2% BSA) or 10 M forskolin to assess the
reactivity of cell preparation. The reaction was stopped by centrifugation (5
min, 4 C,
21
CA 02605036 2007-10-01
12 000 g). Pellets were resuspended in 200 l of lysis buffer (10 min, RT,
vortex)
supplied with the EIA kit (cAMP Direct BiotrakT"" enzyme immunoassay kit,
Amersham Biosciences) and centrifuged (5 min, 4 C, 12 000 g). Supernatents
were
used to quantify immunoreactive cAMP levels (non-acetylated method). Pellets
were
kept frozen for determination of protein content. 59 Optical densities were
measured
at 450 nm, using a microplate reader (Bio-Rad, model 3550). The intra-assay
coefficient of variation was <_ 12% in all experiments. Net GHRH-induced cAMP
levels (without basal level) were expressed in percentage of relative levels
compared
to that obtained in the presence of GHRH.
Quantification of cell proliferation in semi-purified thin limbs of Henle's
loop
cells
Freshly semi-purified cells were cultured in DMEM/F-12 culture media,
containing
antibiotics and the vehicle (culture medium) or 1, 10 or 100 nM rGHRH (1-29)
NH2.
GHRH was added at time 0 and after a 24 and 48 h culture period. Proliferation
was
assessed with aliquots of 40,000 cells, after a 60-h cell culture period,
using the
Promega kit (CeliTiter 96R Aqueous one solution cell proliferation assay).
Data analysis and statistics
RPA represents a more sensitive and reliable method to perform a valid
quantification of GHRH-R mRNA levels in rat renal medulla and Henle's loop
cells
compared to Northern blotting. 33 However, Northern blotting was chosen to
study the
pituitary GHRH-R mRNA levels as it allows the detection of GHRH-R individual
transcripts19. The validity of comparing GHRH-R mRNA levels, using RPA and
Northern blotting was assessed using pituitary total RNA. In the pituitary
from 3-day
water-deprived rats, GHRH-R mRNA levels obtained from Northern blots (sum of
densities of the two transcripts: 3.0 0.2 times higher than controls) were
not
significantly different from those obtained by RPA (sum of densities of the
two
protected fragments: 3.1 0.3 times higher than controls), indicating that
medullary
and pituitary GHRH-R mRNA levels can be compared. Quantification of GHRH-R
mRNA transcripts, protected fragments and visualization of gels were performed
using an IS1000 Digital imaging system (Alpha Innotech Corp/Canberra Packard,
QC).
22
CA 02605036 2007-10-01
Results were expressed as mean SEM. Comparisons of normalized GHRH-R
mRNA levels as well as intracellular cAMP levels, immunofluorescence
intensity,
anti-BrdU immunoreactive cells were performed by ANOVA, followed by the
Dunnett's multiple range test or by the unpaired Student's t test. Statistical
significance of differences was established at P<0.05.
23
CA 02605036 2007-10-01
C
~. =
-H z z
3 N Ln
c ~
0
> o ~ M
+~ Q Q
Q o ~n 00 M N N z z
d U
~
+~+ C
N + p Ga L~
z z
~ 00
L6
L.
O
~ O N
M b H Q Q n
o M 00 N H H
m N rZti Z Z
L U
O
X 09
oo 41 + +
Z M N ~
09
oo c+v -H + N Q -v
00 z ~ ~ =
,
T. N C O
~ Q
u 00 + rn +
O z ~
z M N N n
~-
N
O ~ U C
Rf
C O l- 00 M ~! + O +
U M N M a ~ Z
o a o "
M U
110 N O O
L ~z N~ N N o 0
y- N N O U
0 ~ u
V O~ v1 N N
N 00 N 10 N N v z U
+r ~ ~ o
~ ~ ~ z y o
m
00
U T ~ ~ -6b
m 44
ZI 3
Q) . > x > a'
O z oa w 3 ~E
ZD
CA 02605036 2007-10-01
EXAMPLES
General characteristics of rats submitted to a high-NaCI diet or water
deprivation
Body weight (BW), food and water intakes, urine flow rate and urine sodium
rate of
rats fed a 8%-NaCI diet (GHRH-R mRNA study), and BW and food intake of water-
deprived rats are reported in Table 1. As previously observed, 35, 36 water
intake,
urine flow rate and urine sodium excretion rate of rats submitted to the 8%-
NaCI diet
were significantly increased when compared to controls (P<0.001). After 14-day
of
the high-salt regimen, BW was decreased by 7% (P<0.01). Food intake was not
modified by this diet. No change was observed in BW and food intake of 2-month-
old
rats submitted to a 2-day 8%-NaCI diet and either injected with GHRH or
saline.
BW of water-deprived rats was decreased when compared to controls (P<0.001),
as
reported before. 37 Moreover, their food intake decreased (P<0.001), providing
an
explanation for the loss of BW.
Example 1 GHRH-R Expression Profile
GHRH-R mRNA levels were analyzed by ribonuclease protection assay (RPA). 33
Two distinct bands were detected, using the RPR64 rGHRH-R probe and their sum
was considered as the total level of GHRH-R mRNA, as in previous works. 33, 38
In
kidneys from 2-month-old healthy male rats (Fig. 1), thin limb cells contained
highest
levels of GHRH-R mRNA. Those found in ascending thick limb (ATL) cells and
total
medulla were 5.8 and 3.4 times lower, respectively (P<0.01). GHRH-R mRNA
levels
from ATL cells were 1.7 times lower than those in total medulla (P<0.05).
Immunocytochemical localization of GHRH-R in thin limbs of Henle's loop
As the highest level of GHRH-R mRNA was observed in thin limbs of Henle's loop
(HL) cells, a purified cell preparation was used to assess the precise
localization of
GHRH-R. Co-immunolocalization of GHRH-R with markers of the thin descending
(aquaporin-1) and thin ascending (CIC-K) limbs of HL cells revealed as shown
in Fig.
2 that aquaporin-1 positive cells (Fig. 2a) were devoid of GHRH-R (Figs. 2b,
2c).
However, CIC-K (Fig. 2d) and GHRH-R co-labeling was observed in ~91% of the
cells (Figs. 2e, 2f). No signal was seen when the GHRH-R or CIC-K antibody was
substituted by normal IgGs (data not shown).
CA 02605036 2007-10-01
Immunocytochemical localization and gene expression of GHRH in thin limbs
of Henle's loop
GHRH and CIC-K immunofluorescence was always co-localized as shown in Fig. 3.
No signal was observed when the GHRH primary antibody was substituted by
normal
IgGs (Fig. 3A e). The GHRH fluorescent signal overlapped g:45% of CIC-K
immunoreactive cells (Fig. 3A f). In addition, positive results from RT-PCR
strongly
suggest that immunoreactive preproGHRH is locally synthesized in thin limbs of
HL
(Fig. 3B).
Example 2 In vivo regulation of renal medulla GHRH-R mRNA levels following a
2-, 7- or 14-day high-NaCI diet or a 3- or 5-day water deprivation
As shown in Fig. 4, after a 2- and 7-day 8%-NaCi dietary intake, renal medulla
GHRH-R mRNA levels were 1.4-fold lower (P<0.01) and 1.3-fold higher (P<0.05)
than those of control rats (0.3% NaCi), respectively, when expressed per 20 g
total
RNA, to reflect cellular levels. They were decreased by 1.5-fold after 2 days
(P<0.05)
and increased by 1.7-fold after 7 days (P<0.01) of the 8%-NaCI diet, when
expressed
per medulla total RNA content, to reflect tissue level (data not shown). After
14 days
of the regimen, no significant difference was observed between GHRH-R mRNA
levels from rats submitted to the high-salt diet and controls, either
expressed per 20
g total RNA (Fig. 4 B) or per medulla total RNA content (data not shown).
After 3- or
a 5-day water deprivation, no significant difference was observed between GHRH-
R
mRNA levels from water-deprived and control rats, having free access to water
(data
not shown).
Example 3 In vivo regulation of anterior pituitary GHRH-R mRNA levels
following a 2-, 7- or 14-day high-NaCI diet or a 3- or 5-day water deprivation
The presence of 2.5- and 4-kb GHRH-R mRNA transcripts was observed in the
anterior pituitary of all rats (controls, 8%-NaCl-fed, water-deprived), as
previously
reported. 10 In the pituitary from high-salt-fed rats, no drastic changes of
GHRH-R
mRNA transcript levels were observed when expressed per 12 g total RNA (Fig.
5A-C). After 7 days of the regimen, the levels of the 2.5-kb GHRH-R mRNA
transcript
was transiently decreased by 1.2-fold (P<0.05; Fig. 5B). No change in
pituitary
26
CA 02605036 2007-10-01
GHRH-R mRNA levels was observed, at any time, when data were expressed per
pituitary total RNA content (data not shown).
After 3 days of water deprivation, pituitary levels of the 2.5-kb GHRH-R mRNA
transcript and combined levels of 2.5-kb and 4-kb transcripts, increased 2.8-
and 3.0-
fold (P<0.001), respectively, when expressed per 12 g total RNA (Fig. 5D).
When
GHRH-R mRNA levels were analyzed per anterior pituitary total RNA content,
levels
of the 2.5-kb transcript and combined levels of 2.5-kb and 4-kb transcripts
increased
1.5-fold (P<0.05) (data not shown). After 5 days of water deprivation,
pituitary levels
of the 2.5-kb GHRH-R mRNA transcript and combined levels of 2.5-kb and 4-kb
transcripts, when expressed per 12 g total RNA, increased 1.8- and 1.9-fold
(P<0.001), respectively (data not shown). When GHRH-R mRNA levels were
analyzed per anterior pituitary total RNA content, levels of the 2.5-kb
transcript and
combined levels of 2.5-kb and 4-kb transcripts were increased 1.3- (P<0.05)
and 1.4-
fold (P<0.01), respectively (data not shown).
Example 4: Sensitivity to GHRH in semi-purified thin limbs of Henle's loop
cells
from rats submitted to a 2-, 7- or 14-day high-NaCI diet
Sensitivity to GHRH in thin limbs of Henle's loop cells from rats submitted to
a 2-, 7-
or 14-day high-NaCI diet was assessed by measuring GHRH-induced intracellular
cAMP production, in freshly dispersed semi-purified thin limb cells as shown
in Fig. 6.
Basal or forskolin levels of immunoreactive cAMP were not significantly
decreased in
rats fed 2 days with 8%-NaCl chow, although a trend was observed. Sensitivity
to
rGHRH(1-29)NH2 was altered and GHRH-induced cAMP production was decreased
1.5-fold (1 nM: P<0.01; 100 nM: P<0.05) (Fig. 6A). This loss of sensitivity to
GHRH
was reverted in rats fed the high-salt diet for 7 or 14 days (Fig. 6B, 6C).
Example 5: In vivo effect of a GHRH treatment on DNA repair/synthesis in
purified thin limbs of Henle's loop cells from rats submitted to a 2-day high-
NaCI diet
As shown in Fig. 7, when 2-month-old rats fed 2 days to a 8%-NaCI chow were
injected daily with GHRH (1 mg/kg BW sc/day), a 5 times increase in the number
of
ascending thin limb cell nuclei and mitochondria immunolabeled to BrdU was
observed in thin limbs of Henle's loop cells (P<0.05), when compared to
various
27
CA 02605036 2007-10-01
control groups (normal diet, with or without GHRH injections, high-salt diet
alone).
Moreover, the intensity of mitochondrial BrdU immunofluorescence was increased
'Z~7
times in these cells (P<0.05). GHRH-R mRNA levels tended to increase in the
renal
medulla of the high-NaCI fed rats, injected with GHRH and serum total insulin-
like
growth factor-1 (IGF-1) levels were not modified (data not shown).
Example 6: In vivo effect of a GHRH treatment on GHRH-R and CICK-1 mRNA
levels in purified thin limb cells from rats submitted to a 2-day high-NaCI
diet
GHRH-R (Fig. 8A) and CICK-1(Fig. 8B) mRNA levels, measured by real-time RT-
PCR, were significantly increased in the total renal medulla of a subgroup of
3 rats.
GHRH-R mRNA levels were decreased without significantly altering those of CIC-
K1
in 5 others. These results indicate that a lower GHRH dosage, such as 0.5
mg/kg
BW/day, will up-regulate the renal GHRH-R in a large number of rats.
Example 7: RT-PCR products from rat and porcine anterior pituitary and renal
medulla, using a panel of anterior pituitary GHRH-R primers
Using primers targeting the 5' end, median portion and 3' end of the pituitary
GHRH-
R, a similar pattern was observed in both the rat and porcine renal medulla in
comparison with anterior pituitary. No signal was detected in the rat (Fig. 9
A) and
porcine (Fig. 9B) medulla when 5' end primers were used.
Example 8: In vivo effect of a GHRH treatment on cell proliferation in
purified
thin limb cells from normal rats
As shown in Fig. 10, the effect of GHRH on proliferation was directly assessed
in
semi-purified thin limbs of Henle's loop cells from healthy 2-month-old rats.
rGHRH(1-
29)NH2 induced a 2.4 to 3.2-fold increase of the proliferative index in these
cells (1
and 10 nM: P<0.05; 100 nM: P<0.01) when compared to control cell stimulated
with
the GHRH vehicle.
28
CA 02605036 2007-10-01
The invention being herein above described, it will be obvious that the same
may be
varied in many ways. Those skilled in the art recognize that other and further
changes and modifications may be made thereto without departing from the
spirit of
the invention, and it is intended that all such changes and modifications fall
within the
scope of the invention, as defined in the appended claims.
29
CA 02605036 2007-10-01
SEQUENCE LISTING
SEQ ID NO. :1- GHRH(1-44)
Tyr Ala Asp Ala Ile Phe Thr Asn Ser Tyr Arg Lys Val Leu Gly Gln Leu Ser Ala
Arg Lys
Leu Leu Gln Asp Ile Met Ser Arg Gln GIn Gly Glu Ser Asn Gln Glu Arg Gly Ala
Arg
Ala Arg Leu
SEQ ID NO.2:
Tyr Ala Asp Ala Ile Phe Thr Asn Ser Tyr Arg Lys Val Leu Gly Gln Leu Ser Ala
Arg Lys
Leu Leu Gln Asp Ile Met Ser Arg-Xaa
Wherein Xaa is either absent or any amino acid sequence of 1 up to 15 residues
SEQ ID NO.:3 - GHRH(1-29)
Tyr Ala Asp Ala Ile Phe Thr Asn Ser Tyr Arg Lys Val Leu Gly Gln Leu Ser Ala
Arg Lys
Leu Leu Gln Asp Ile Met Ser Arg
SEQ ID NO. :4
Tyr-D-Ala2-Asp-Ala-I Ie-Phe-Thr-Ala-Ser-Tyr-Arg-Lys-Val-Leu-Ala-Gln-Leu-Ser-
Ala-
Arg-Lys-Lys-Leu-Gln-Asp-I Ie-Met-Ser-Arg-Xaa,
wherein Xaa is either absent or any amino acid sequence of 1 up to 15 residues
SEQ ID NO. :5
Tyr-D-Ala2-Asp-Ala-Ile-Phe-Thr-Asn-Ser-D-Tyr90-Arg-Lys-Val-Leu-Gly-Gln-Leu-Ser-
AIa-Arg-Lys-Lys-Leu-G I n-Asp-I le-Met-Ser-Arg-Xaa
wherein Xaa is either absent or any amino acid sequence of 1 up to 15 residues
SEQ ID NO. :6
Tyr- D-AIaz-Asp-AIa-IIe-Phe-Thr-Asn-Ser-D-Tyr10-Arg-Lys-Val-Leu-D-Ala'5-Gln-
Leu-
Ser-Ala-Arg-Lys-Lys22-Leu-Gln-Asp-I Ie-Met-Ser-Arg-Xaa
wherein Xaa is either absent or any amino acid sequence of 1 up to 15 residues
SEQ ID NO.:7
Tyr Ala Asp Ala Ile Phe Thr Ala 8 Ser Tyr Arg Lys Val Leu Ala15 Gin Leu Ser
Ala Arg
Lys Ala22 Leu Gln Asp Ile Met Ser Arg
CA 02605036 2007-10-01
SEQ ID NO. :8
Tyr Ala Asp Ala Ile Phe Thr Ala 8 Ala9 Tyr Arg Lys Val Leu Ala15 Gln Leu Ser
Ala Arg
Lys Ala22 Leu Gln Asp Ile Met Ser Arg
SEQ ID NO. :9
Tyr Ala Asp Ala Ile Phe Thr Asn Ser Tyr Arg Lys Val Leu Gly Gln Leu Ser Ala
Arg Lys
Lys22 Leu Gln Asp Ile Met Ser Arg
SEQ ID NO.:10
Tyr-Xaa2-Asp-Ala-Ile-Phe-Thr-Xaa8-Xaa9-Xaa10-Arg-Lys-Val-Leu-Xaa 15-Gln-Leu-
Ser-Ala-Arg-Xaa2l-Xaa22-Leu-Gln-Asp-I le-Met-Ser-Arg-Xaa30,
wherein :
Xaa2 is Ala or D-Ala;
Xaa 8 is Asn, D-Asn or Ala;
Xaa 9 is Ser or Ala;
Xaa 10 is Tyr or D-Tyr;
Xaa 15 is Gly, Ala or D-Ala;
Xaa 21 is Lys or D-Lys;
Xaa 22 is Leu, D-Leu, Lys or Ala; and
Xaa 30 is a bond or any amino acid sequence of 1 up to 15 residues and wherein
the
analogue comprises at least one of the above amino acid substitution in
comparison
with the amino acid sequence of the native form of hGHRH1-29.
31
CA 02605036 2007-10-01
REFERENCES
1. Lin C, Lin SC, Chang CP et al. Pit-1 dependent expression of the receptor
for
growth hormone-releasing factor mediates pituitary cell growth. Nature 1992;
360:765-768.
2. Mayo KE. 1992 Molecular cloning and expression of a pituitary specific
receptor
for growth hormone-releasing factor. Mol Endocrinol 1992; 6:1734-1744.
3. Hsiung HM, Smith DP, Zhang XY et al. Structure and functional expression of
a
complementary DNA for porcine growth hormone-releasing hormone receptor.
Neuropeptides 1993; 25:1-10.
4. Horikawa R, Gaylinn BD, Lyons Jr CE et al. Molecular cloning of ovine and
bovine growth hormone-releasing hormone receptors: the ovine receptor is C-
terminally truncated. Endocrinology 2001; 142:2660-2668.
5. Hashimoto K, Koga M, Motomura T et al. Identification of alternatively
spliced
messenger ribonucleic acid encoding truncated growth hormone-releasing hormone
receptor in human pituitary adenomas. J Clin Endocrinol Metab 1995; 80:2933-
2939.
6. Tang J, Lagace G, Castagne J et al. Identification of human growth hormone-
releasing hormone receptor splicing variants. J Clin Endocrinol Metab 1995;
80:2381-
2387.
7. Gaylinn BD, Harrison JK, Zysk JR et al. Molecular cloning and expression of
human anterior pituitary receptor for growth hormone-releasing hormone. Mol
Endocrinol 1993; 7:77-84.
8. Toogood AA, Harvey S, Thorner MO et al. Cloning of the chicken pituitary
receptor for growth hormone-releasing hormone. Endocrinology 2006; 147:1838-
1846.
9. Lee LT, Sju FK, Lau IT et al. Discovery of growth hormone-releasing
hormones
and receptors in nonmammalian vertebrates. Proc Natl Acad Sci USA
2007;104:2133-2138.
10. Girard N, Boulanger L, Denis S et al. Differential in vivo regulation of
the pituitary
growth hormone-releasing hormone (GHRH) receptor by GHRH in young and aged
rats. Endocrinology 1999; 140:2836-2842.
11. Miller TL, Godfrey PA, Dealmeida VI et al. The rat growth hormone-
releasing
hormone receptor gene: structure, regulation, and generation of receptor
isoforms
with different signaling properties. Endocrinology 1999; 140:4152-4165.
32
CA 02605036 2007-10-01
12. Barinaga M, Bilezikjian LM, Vale WW et al. Independent effects of growth
hormone releasing factor on growth hormone release and gene transcription.
Nature
1985; 314:279-281.
13. Tannenbaum GS, Ling N. The interrelationship of growth hormone (GH)-
releasing factor and somatostatin in generation of the ultradian rhythm of GH
secretion. Endocrinology 1984; 115:1952-1957.
14. Bilezikjian LM, Vale WW. Stimulation of adenosine 3',5'-monophosphate
production by growth hormone-releasing factor and its inhibition by
somatostatin in
anterior pituitary cells in vitro. Endocrinology 1983; 113:1726-1731.
15. Cuttler L, Glaum SR, Collins BA et al. Calcium signaling in single growth
hormone-releasing factor-responsive pituitary cells. Endocrinology 1992;
130:945-
953.
16. Lussier BT, French MB, Moore BC et al. Free intracellular Ca2+
concentration
([Ca2+];) and growth hormone release from purified rat somatotrophs. I. GH-
releasing factor-induced Ca2+ influx raises [Ca2+];. Endocrinology 1991;
128:570-582.
17. Lussier BT, French MB, Moore BC et al. Free intracellular Caz+
concentration
and growth hormone (GH) release from purified rat somototrophs. III. Mechanism
of
action of GH-releasing factor and somatostatin. Endocrinology 1991; 128:592-
603.
18. Chen C, Xu R, Clarke IJ et al. Diverse intracellular signaling systems
used by
growth hormone-releasing hormone in regulating voltage-gated Ca2+ or K
channels
in pituitary somatotropes. Immunol Cell Biol 2000; 78: 356-368.
19. Billestrup N, Swanson LW, Vale W. Growth hormone-releasing factor
stimulates
proliferation of somatotrophs in vitro. Proc Natl Acad Sci USA 19986; 83:6854-
6857.
20. Dean CE, Porter TE. Regulation of somatotroph differentiation and growth
hormone (GH) secretion by corticosterone and GH-releasing hormone during
embryonic development. Endocrinology 1999; 140:1104-1110.
21. Godfrey P, Rahal JO, Beamer WG et al. GHRH receptor of little mice
contains a
missence mutation in the extracellular domain that disrupts receptor function.
Nat
Genet 1993; 4:227-232.
22. Lin SC, Lin CR, Gukovsky I. Molecular basis of the little mouse phenotype
and
implication for cell type-specific growth. Nature 1993; 364:208-213.
23. Pombo CM, Zalvide J, Gaylinn BD et al. Growth hormone-releasing hormone
stimulates mitogen-activated protein kinase. Endocrinology 2000; 141:2113-
2119.
33
CA 02605036 2007-10-01
24. Zeitler P, Siriwardana G. Stimulation of mitogen-activated protein kinase
pathway in rat somatotrophs by growth hormone-releasing hormone. Endocrine
2000; 12:257-264.
25. Dickson PR, Feifel D, Vaccarino FJ. Blockade of endogenous GRF at dark
onset
selectively suppresses protein intake. Peptides 1995; 16:7-9.
26. Zhang J, Obal F Jr, Zheng T et al. Intrapreoptic microinjection of GHRH or
its
antagonist alters sleep in rats.J Neurosci 1999; 19:2187-2194.
27. Guarcello V, Weigent DA, Blalock JE. Growth hormone-releasing hormone
receptors on thymocytes and splenocytes from rats. Cell Immunol 1991; 136:291-
302.
28. Gallego R, Pintos E, Garcia-Caballero T et al. Cellular distribution of
growth
hormone-releasing hormone receptor in human reproductive system and breast and
prostate cancers. Histol Histopathol 2005; 20:697-706.
29. Kotani E, Usuki S, Kubo T. Effect of growth hormone-releasing hormone on
luteinizing hormone stimulated progestin biosynthesis in cultured rat ovarian
granulosa cells. Gynecol Endocrinol. 1998; 12:307-313.
30. Moretti C, Bagnato A, Solan N er al. Receptor-mediated actions of growth
hormone-releasing factor on granulosa cell differentiation. Endocrinology
1990;
127:2117-2126.
31. Margioris AN, Brockmann G, Bohler HC et al. Expression and localization of
growth hormone-releasing hormone messenger ribonucleic acid in rat placenta:
in
vitro secretion and regulation of its peptide product. Endocrinology 1990;
126:151-
158.
32. Ciampani T, Fabbri A, Isidori A et a/. Growth hormone-releasing hormone is
produced by rat Leydig cell in culture and acts as a positive regulator of
Leydig cell
function. Endocrinology 1992; 131:2785-2792.
33. Boisvert C, Pare C, Veyrat-Durebex C et al. Localization and regulation of
a
functional GHRH receptor in the rat renal medulla. Endocrinology 2002;
143:1475-
1484.
34. Boulanger L, Girard N, Strecko J et al. Characterization of a growth
hormone-
releasing hormone binding site in the rat renal medulla. Peptides 2002;
23:1187-
1194.
35. Ying WZ, Sanders PW. Dietary salt enhances glomerular endothelial nitric
oxide
synthase through TGF-betal. Am J Physiol 1998; 275:F18-F24.
34
CA 02605036 2007-10-01
36. Ying WZ, Sanders PW. Dietary salt regulates expression of Tamm-Horsfall
glycoprotein in rats. Kidney Int 1998; 54:1150-1156.
37. Shin SJ, Wen JD, Chen IH et al. Increased renal ANP synthesis, but
decreased
or unchanged cardiac ANP synthesis in water-deprived and salt-restricted rats.
Kidney Int 1998; 54:1617-1625.
38. Korytko Al, Zeitler P, Cuttler L. Developmental regulation of pituitary
growth
hormone-releasing hormone receptor gene expression in the rat. Endocrinology
1996; 137:1326-1331.
39. Matsubara S, Sato M, Mizobuchi M et al. Differential gene expression of
growth
hormone (GH)-releasing hormone (GRH) and GRH receptor in various rat tissues.
Endocrinology 1995; 136:4147-4150.
40. Fujinaka Y, Yokogoshi Y, Zhang CY et al. Tissue-specific molecular
heterogeneity of human growth hormone-releasing hormone receptor protein. FEBS
Lett 1996; 9394:1-4.
41. Nielsen S, Pallone T, Smith BL et al. Aquaporin-1 water channels in short
and
long loop descending thin limbs and in descending vasa recta in rat kidney. Am
J
Physiol 1995; 268:F1023-1037
42. Uchida S, Sasaki S, Nitta K et al. Localization and functional
characterization of
rat kidney-specific chloride channel, CIC-K1. J Clin Invest 1995; 95:104-113.
43. Kondo Y, Kudo K, Igarashi Y. Functions of ascending thin limb of Henle's
loop
with special emphasis on mechanism of NaCI transport. Tohoku J Exp Med 1992;
166:75-84.
44. Rodriguez-Iturbe B, Sepassi L, Quiroz Y. Association of mitochondrial SOD
deficiency with salt-sensitive hypertension and accelerated renal senescence.
J Appl Physiol 2007; 102:255-260.
45. Taylor NE, Cowley AW. Effect of renal medullary H202 on salt-induced
hypertension and renal injury. Am J Physiol Regul lntegr Comp Physiol 2005;
289:R1573-R1579.
46. Liang M, Yuan B, Rute E et al. Insights into Dahi salt-sensitive
hypertension
revealed by temporal patterns of renal medullary gene expression. Physiol
Genomics
2003; 12:229-237.
47. Hisaki R, Fujita H, Saito F. Tempol attenuates the development of
hypertensive
renal injury in Dahi salt-sensitive rats. Am J Hypertens 2005; 18:707-713.
CA 02605036 2007-10-01
48. Zhang JJ, Bledsoe G, Kato K. Tissue kallikrein attenuates salt-induced
renal
fibrosis by inhibition of oxidative stress. Kidney Int 2004; 66:722-732.
49. Lee H, Wei YH Oxidative stress, mitochondrial DNA mutation and apoptosis
in
aging.Exp Biol Med 232:592-606. Tan AL, Forbes JM, Copper ME 2007 AGE, RAGE,
and ROS in diabetic nephropathy. Semin Nephrol 2007; 27:130-143.
50. Favier A 2006 Oxidative stress in human diseases Ann Pharm Fr 2006; 64:390-
396.
51. Csiszar A, Toth J, Petri-Peterdi J, Ungvari Z The aging kidney: role of
endothelial oxidative stress and inflammation. Acta Physiol Hung 2007; 94:107-
115.
52. Morel G, Gallego R, Boulanger L et al. Restricted presence of the growth
hormone-releasing hormone receptor to somatotrophs in rat and human
pituitaries.
Neuroendocrinology 1999; 70:128-136.
53. Bruno FH, Sing J, Berelowitz M. Regulation of rat hypothalamic
preprogrowth
hormone-releasing factor messenger ribonucleic acid by dietary protein.
Endocrinology 1991; 129:1226-1232.
54. Zeitler P, Stevens P, Siriwardana G. Functional GHRH receptor carboxyl
terminal isoforms in normal and dwarf (dw) rats. J Mol Endocrinol 1988; 21:363-
371.
55. Grupp C, Lottermoser J, Cohen DI et al. Transformation of rat inner
medullary
fibroblasts to myofibroblasts in vitro. Kidney Int 1997; 52:1279-1290.
56. Gaudreau P, Boulanger L, Abribat T. Affinity of human growth hormone-
releasing factor (1-29)NH2 analogues for GRF binding sites in rat
adenopituitary. J
Med Chem. 1992; 35:1864-1869.
57. Trinh-Trang-Tan MM, Bouby N, Coutaud C et al. Quick isolation of rat
medullary
ascending limbs. Pflugers Arch 1986; 407:228-234.
58. Vetrat-Durebex C, Pomerleau L, Langlois D et al. Internalization and
trafficking
of the human and rat growth hormone-releasing hormone receptor. J Cell Physiol
2005; 203:335-344.
59. Bradford M. A Rapid and sensitive method for the quantitation of microgram
quantities of protein utilizing the principle of protein-dye binding. Anal
Biochem 1976;
72:248-254.
60. Mirescu et al., Global assessment of serum antioxidant status in
hemodialysis
patients. J Nephrol. 2005;18:599-605.
36
CA 02605036 2007-10-01
61. Agarwal R and Chase SD. Rapid, fluorometric-liquid chromatographic
determination of malondialdehyde in biological samples. J of Chromatography B
2002; 775: 121-126.
62. Aebi H, Catalase in vitro, Methods Enzymol 1984; 105:121-6.
63. Paglia DE, Valetine WN., Studies on the quantitative and qualitative
characterization of erythrocyte glutathione peroxidase. J. Lab Clin. Invest.
1976;70 :158-69
64. Carlberg I. and B. Mannervik, Glutathione reductase, Methods Enzymol. 13
(1985), pp. 484-499.
65. Arthur J.R. Functional indicators of iodine and selenium status. Proc Nutr
Soc.
1999;58:507-12.
66. Reznick AZ, Packer L. Oxidative damage to proteins : spectrophotometric
method for carbonyl assay. Methods Enzymol, 1994;233:357-63.
37
DEMANDES OU BREVETS VOLUMINEUX
LA PRESENTE PARTIE DE CETTE DEMANDE OU CE BREVETS
COMPREND PLUS D'UN TOME.
CECI EST LE TOME 1 DE 2
NOTE: Pour les tomes additionels, veillez contacter le Bureau Canadien des
Brevets.
JUMBO APPLICATIONS / PATENTS
THIS SECTION OF THE APPLICATION / PATENT CONTAINS MORE
THAN ONE VOLUME.
THIS IS VOLUME 1 OF 2
NOTE: For additional volumes please contact the Canadian Patent Office.
