CA2605036A1 - Renoprotection by growth hormone-releasing hormone and agonists - Google Patents

Abstract

In the present study, regulation of renal and pituitary GHRH-R mRNA levels was examined using in vivo models of NaCl or water homeostasis disruption. The presence of a unique GHRH/GHRH-R system in Henle's loop ascending thin limb cell and the specific regulation of GHRH-R mRNA levels and GHRH sensitivity in a situation of hyperosmotic stress, together with the strong effect of GHRH on mitochondrial and nuclear DNA repair/synthesis, indicate a role for GHRH in renoprotection. GHRH appears to be involved in adaptive processes related to DNA
repair and/or synthesis thereby protecting ascending limb cell function in subjects with renal vulnerability (aging, diabetes) and were a health event could lead to a production of oxidative stress (antibiotic toxicity, cancer chemotherapeutic agent toxicity, infection, inflammation, ischemia ) and renal failure.

Description

DEMANDES OU BREVETS VOLUMINEUX
LA PRESENTE PARTIE I)E CETTE DEMANDE OU CE BREVETS
COMPREND PLUS D'UN TOME.
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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 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 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.

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 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-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 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

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 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 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-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).

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.

*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 Aqueous one solution cell proliferation assay. Results represent the mean SEM of 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 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 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 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 (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 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 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 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).

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 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 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, 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).

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.

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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).

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 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 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-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.

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.

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 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.

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Claims (24)

1. A method of preventing the death of kidney cells and/or loss of kidney function due to oxidative stress in a mammal in need thereof, the method comprising administering a ligand to the GHRH renal receptor to the mammal.

2. The method of claim 1, wherein said ligand is GHRH, a biologically active fragment of GHRH or a GHRH agonist thereof.

3. The method of claim 2, wherein said ligand selected from the group consisting of SEQ ID NO.:1, SEQ ID NO.:2 and SEQ ID NO.:3.

4. The method of claim 2, wherein said ligand is selected from the group consisting of SEQ ID NO.:4, SEQ ID NO.:5, SEQ ID NO.:6, SEQ ID NO.:7, SEQ ID NO.:8, SEQ ID NO.:9 and SEQ ID NO.:10.

5. The method of any one of claims 1 to 4, comprising identifying the mammal in need by determing the presence of marker associated with an oxidative stress to renal cells.

6. The method of any one of claims 1 to 5, wherein the mammal suffers or is susceptible of suffering from a disease selected from the group consisting nephropathy, diabetes insipidus, diabetes type I, diabetes II, renal disease glomerulonephritis, bacterial or viral glomerulonephritides, IgA nephropathy, Henoch-Schonlein Purpura, membranoproliferative glomerulonephritis, membranous nephropathy, Sjogren's syndrome, nephrotic syndrome minimal change disease, focal glomeruloscierosis 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, tuborous 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 crecentic glomerulonephritis, polymyositis-dermatomyositis, pancreatitis, rheumatoid arthritis, systemic lupus erythematosus, gout, blood disorders, sickle cell disease, thrombotic thrombocytopenia purpura, hemolytic-uremic syndrome, acute corticol necrosis, renal thromboembolism, trauma and surgery, extensive injury, burns, abdominal and vascular surgery, induction of anesthesia, side effect of use of drugs or drug abuse, 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, soriasis, systemic sclerosis, respiratory disease, COPD, obstructive sleep apnoea, hypoia at high altitude or erdocrine disease, acromegaly, diabetes mellitus, diabetes insipidus, and conditions related to antibiotic toxicity, infection, inflammation, ischemia.

7. The method of any one of claims 1 to 5, wherein the mammal is subjected to chronic hemodialysis.

8. A method of promoting regeneration of kidney cells and/or kidney function in a mammal in need thereof, the method comprising administering a ligand to the GHRH renal receptor to the mammal.

9. The method of claim 8, comprising identifying the mammal in need by determing the presence of a marker associated with an oxidative stress to renal cells.

10.The use of an effective amount of a ligand to the GHRH renal receptor to protect a subject against oxidative renal damage.

11.The use as defined in claim 10, wherein said ligand is a GHRH agonist capable of activating and upregulating renal GHRH receptor.

12. The use as defined in claims 10 or 11, wherein said ligand is GHRH(1-29)NH2.

13.The use as defined in any one of claims 10-12, wherein said effective amount of ligand is not substantially active against anterior pituitary GHRH
receptor.

14.The use as defined in claim 13, wherein this effective amount has the same protective effect as a subcutaneous 1.0 mg rat GHRH(1-29)NH2 dose per kilogram of body weight per day or lower, in a Sprague Dawley rat submitted to a high-salt diet.

15.The use as defined in any one of claims 10 to 14, wherein said oxidative renal damage affects ascending thin limb Henle's loop epithelial cells.

16. The use of claim 15, wherein said oxidative damage is due to exaggerated renal medullary osmolality.

17.The use a ligand to the GHRH renal receptor for preventing the death of kidney cells and/or loss of kidney function due to oxidative stress.

18.The use as defined in claim 17, wherein said ligand is GHRH, a biologically active fragment of GHRH or a GHRH agonist thereof.

19.The use as defined in claim 18, wherein said ligand is selected from the group consisting of SEQ ID NO.:1, SEQ ID NO.:2 and SEQ ID NO.:3.

20.The use as defined in claim 18, wherein said ligand is selected from the group consisting of SEQ ID NO.:4, SEQ ID NO.:5, SEQ ID NO.:6, SEQ ID NO.:7, SEQ ID NO.:B, SEQ ID NO.:9 and SEQ ID NO.:10.

21.The use as defined in any one of claims 17 to 20, wherein the mammal suffers or is susceptible of suffering from a disease selected from the group consisting nephropathy, diabetes insipidus, diabetes type I, diabetes II, renal disease glomerulonephritis, bacterial or viral glomerulonephritides, 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, tuborous 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 crecentic glomerulonephritis, polymyositis-dermatomyositis, pancreatitis, rheumatoid arthritis, systemic lupus erythematosus, gout, blood disorders, sickle cell disease, thrombotic thrombocytopenia purpura, hemolytic-uremic syndrome, acute corticol necrosis, renal thromboembolism, trauma and surgery, extensive injury, burns, abdominal and vascular surgery, induction of anesthesia, side effect of use of drugs or drug abuse, 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, soriasis, systemic sclerosis, respiratory disease, COPD, obstructive sleep apnoea, hypoia at high altitude or erdocrine disease, acromegaly, diabetes mellitus, diabetes insipidus, and conditions related to antibiotic toxicity, infection, inflammation, ischemia.

22.The use as defined in any one of claims 17 to 20, wherein the mammal is subjected to chronic hemodialysis.

23.The use of a a ligand to the GHRH renal receptor for promoting regeneration of kidney cells and/or function in a mammal in need thereof.

24.The use as defined in any one of claims 17 to 23, comprising identifying the mammal in need by determing the presence of marker associated with an oxidative stress to renal cells.