Expression of androgen receptor and androgen regulation of NDRG2 in the rat renal collecting duct
Androgens are known to regulate gene expression in the renal proximal tubule. Whether the distal parts of the nephron, in particular the cortical collecting duct (CCD), where sodium reabsorption is controlled tightly by aldosterone, are also targets for these hormones is unknown. Real-time PCR on rat isolated renal tubules showed that androgen receptor mRNA is not only, as expected, expressed in the proximal tubule, but also in the CCD. We examined the effects of adrenalectomy (ADX) plus castration and in-vivo administration of the active metabolite of testosterone, dihydrotestosterone (DHT), on the intrarenal expression of N-myc downstream regulated gene 2 (NDRG2), an early aldosterone-induced gene located specifically in the CCD. NDRG2 belongs to a newly identified family of differentiation-related genes; although the function of these genes remains elusive, regulation of NDRG1 by androgens has been suggested. Castration plus ADX increased NDRG2 expression (RNase protection assay) significantly in the whole kidney, and a single i.p. injection of DHT caused a significant decrease in NDRG2 expression 4 h afterwards (up to 24 h). Furthermore, real-time PCR on microdissected tubules revealed that the decrease in NDRG2 expression caused by DHT is restricted to the CCD. Thus, aldosterone and androgens have opposite effects on NDRG2 expression in the renal CCD. These results are the first demonstration of androgen-dependent gene regulation in the rat renal CCD.
KeywordsDihydrotestosterone NDRG2 Steroid hormones Aldosterone
Androgens exert effects on many tissues, but their major target is defined classically as the male reproductive tract, where they play an important role in the development of the embryonic reproductive system and in the control of secondary sexual characteristics . Androgens also act on a variety of organs, including kidney proximal tubule, liver, cardiac muscle and pituitary gland . The main action of androgens is the stimulation of cell growth including hypertrophy and hyperplasia. In the kidney, androgens play a role in the maturation of kidney in males , but their effects on renal function are unknown. Ex-vivo studies have indicated that dihydrotestosterone, the main metabolite of testosterone, stimulates DNA synthesis of cultured mouse proximal convoluted tubules (PCT) and straight proximal tubules (PR) . Androgens have also been shown to regulate the expression of a variety of genes expressed in proximal tubule cells of rat and mouse kidneys [1, 2, 4], such as ornithine decarboxylase (ODC), an enzyme involved in polyamine synthesis; alcohol dehydrogenase-1 (ADH-1), an enzyme of alcohol metabolism; β-glucuronidase (GUS) involved in the metabolism of glucuronides; the ubiquitously expressed renal protein 2 (RP-2) gene or the kidney androgen-regulated gene (KAP), highly expressed in proximal tubule cells and interacting with the cyclosporine A-binding protein cyclophilin B .
In their target tissues, androgens act by binding to the androgen receptor (AR), which belongs to the nuclear receptor superfamily . The hormone binds to its receptor and the complex translocates to the nucleus where it interacts with glucocorticoid response elements located in the promoter region of regulated genes; this leads to their transcriptional induction or repression. The precise, cell-specific expression of AR within the kidney, apart its high expression in proximal tubule cells, still remains poorly documented . Because androgens exert pleiotropic effects, the question arises to know whether they could also have, in addition to their action on a variety of androgen-regulated genes expressed in proximal tubule cells, some regulatory effects on gene(s) expressed in the more distal segments of the renal tubule, where the mineralocorticoid receptor (MR) is specifically located .
Aldosterone plays a major role in the regulation of sodium homeostasis and blood pressure [7, 8]. In the kidney, it binds to the mineralocorticoid receptor (MR, another member of the nuclear receptor superfamily) in the collecting duct and initiates a cascade of events leading to the induction (or repression) of genes, referred to as aldosterone-induced or -repressed proteins, which are responsible for an increase in sodium reabsorption. In recent years, many efforts have been devoted to the identification of the primary aldosterone-induced or -repressed protein(s) [9, 10, 11, 12]. Recently, we have identified NDRG2, a member of the N-myc downstream-regulated gene (NDRG) gene family, as an early aldosterone-response gene in the rat kidney and cultured rat cortical collecting duct (CCD) cells . The function of the differentiation-related NDRGs, consisting of four members (NDRG1-4) is still largely unknown. Ling and Chang  first identified an early-responsive androgen target gene, referred to as TTD5 or later as NDRG1, that is repressed preferentially by testosterone in 5α-reductase-deficient T-cell hybridoma cells. NDRG1, in contrast, is up-regulated markedly by androgens in prostatic adenocarcinoma cells . The question arises as to whether androgens may also regulate NDRG2 in the kidney. Because the intrarenal distribution of AR is not known, we first investigated the precise localization of AR within the kidney. We then examined the effect of dihydrotestosterone (DHT) on the expression of NDRG2 in whole kidneys and isolated microdissected tubule segments of adrenalectomized, castrated rats.
This study is the first demonstration of the presence of AR mRNA in the cortical collecting duct (CCD) and shows that DHT down-regulates the expression of NDRG2 in this nephron segment, an effect opposite to that of aldosterone. These results open new insights on possible complementary androgenic and mineralocorticoid hormonal regulation of genes expressed in the CCD.
Materials and methods
Plasma levels of aldosterone and testosterone in normal and castrated and adrenalectomized (ADX) rats. Means±SE from blood samples of three different normal rats, four castrated rats and four castrated rats 7 days after ADX
Castration + ADX
Microdissected renal tubules
Various segments of the renal tubule, including proximal convoluted tubule (PCT), straight proximal tubule or pars recta (PR), medullary (mTAL) and cortical (cTAL) parts of the thick ascending limb of Henle’s loop and the CCD were microdissected from rat kidneys as previously described . After perfusion with collagenase (0.1%; Serva France), the kidneys were removed rapidly and thin pyramid-shaped pieces containing cortex and medulla were incubated in a solution (DMEM/HAM’s F12 1:1; 14 mM NaHCO3; 2 mM glutamine; 10 U/ml penicillin-streptomycin; 20 mM HEPES pH 7.4) containing 0.1% collagenase for 60 min at 30°C. The pyramids were then rinsed in the same ice-cold incubation solution without collagenase. Thereafter, tubule segments were isolated under stereomicroscopic observation with the aid of thin needles at 4°C. In each experiment, isolated tubules were measured and pooled into a single sample (PCT, 20–40 mm; PR, 15–25 mm; mTAL, 10–25 mm; cTAL, 25–40 mm; CCD, 17–30 mm); in some experiments, it was possible to collect up to four samples of the same category of tubules; samples were transferred in 175 μl lysis solution, and then stored at −80°C before use.
Real-time quantitative PCR
Total RNA was extracted from renal tubules and treated with DNase I using the SV Total RNA isolation System kit (Promega, Madison, Wisc., USA). RNA was first reverse transcribed using Superscript II reverse transcriptase (Invitrogen, Cergy Pontoise, France) and 500 ng random hexamer (Amersham Biosciences Europe, Orsay, France) in a final volume of 20 μl. Real-time PCR analyses of NDRG2 and AR were carried out on a ABI7700 Sequence Detector (Applied Biosystems, Foster City, Calif., USA). Taqman probe and primers (MWG Biotech) for NDRG2 were as follows: Taqman probe, 5′-FAM (6-carboxyfluorescein)-AACAGTGCCTTGACGTTTAAGGCCTCTGA-TAMRA (6-carboxytetramethylrhodamine)-3′; upper primer, 5′-TCCATTCCCCCAAAGCTG-3′; lower primer, 5′-CATCCATTTAGGAGCATTGCC-3′. Taqman probe and primers for AR were as follows: Taqman probe, 5′-FAM-CCTTGCCTGGCTTCCGCAACTTG-TAMRA-3′; upper primer, 5′-TGTGGTCAAGTGGGCCAAG-3′; lower primer, 5′-TGCCATCTGGTCATCCACAT-3′. The primers for 18S RNA, used as internal standard, were: upper primer, 5′-CCCTGCCCTTTGTACACACC-3′; lower primer, 5′-CGATCCGAGGGCCTCACTA-3′; and Taqman probe, 5′-FAM-CCCGTCGCTACTACCGATTGGATGGT-TAMRA-3′. PCR was performed with 1/20th of the reverse-transcription reaction, with 5 mM (for NDRG2 and AR) or 3 mM (for 18S RNA) MgCl2, 200 μM dNTPs and 1.25 units of Taq polymerase. The concentrations of the primers and probes used were 400 and 100 nM, respectively. PCR reagents were from the qPCR core kit (Eurogentec, Seraing, Belgium). After an initial denaturation step for 10 min at 95°C, the thermal cycling conditions were 40 cycles at 95°C for 15 s and 60°C for 1 min. Standard curves were generated using serial dilutions of a purified fragment of NDRG2 (nt 88–1209), AR (nt 1892–2251), and 18S RNA (nt 1428–1788), covering five orders of magnitude and yielding correlation coefficients of at least 0.98. Each standard and sample values were determined from triplicate measurements. NDRG2 and AR mRNA expression were calculated relative to the 18S RNA.
RNase protection assay
Total RNA was extracted from rat kidney with Trizol reagent (Gibco-BRL) according to the manufacturer’s instructions. RNase protection assay (RPA) using 32P-labelled cRNA probes was performed as previously described [13, 19]. The NDRG2 cRNA probe was 573 nt long (the protected fragment was 447 nt long: 1500–1946), the rat KAP cRNA probe was 379 nt long (the protected fragment was 326 nt long: 356–682), and the rat GAPDH cRNA probe, used as control, was 183 nt long (the protected fragment was 164 nt long: 707–871).
Values are means±SEM from at least three independent experiments. The significance of differences between groups was determined by ANOVA and Student’s t-test. P<0.05 was considered significant.
Androgen receptor mRNA expression along the renal tubule of rat kidney
NDRG2 mRNA expression along the renal tubule
NDRG2 mRNA expression along the rat nephron. NDRG2 mRNA was quantified in tubular samples by real-time PCR. Tubules were microdissected from four rats. PCT proximal convoluted tubules, PR pars recta, mTAL, cTAL medullary and cortical parts respectively of the thick ascending limb of Henle’s loop, CCD cortical collecting ducts. Pools of 16–40 mm of each tubular segment were collected in each of the four rats. Means±SEM
NDRG2/18S mRNA relative expression (arbitrary units)
NDRG2 and KAP mRNA expression in whole kidneys following castration and DHT injection
In this study, we show that the rat CCD expresses the AR and that NDRG2 is an androgen-regulated gene that is almost completely repressed upon DHT treatment. NDRG2 belongs to a recently identified family of differentiation-related genes that consists of four members (NDRG1-4) . NDRGs are highly conserved through evolution and among species, suggesting that they exert important biological function(s). NDRG1 has been characterised as a novel marker of androgen-induced differentiation in the human prostate  and as an early-response androgen target gene differentially down-regulated by androgens in cultured T-cell hybridoma . NDRG1 is also up-regulated in differentiated colonic cells and down-regulated in tumour cells [21, 22]. NDRG3 and NDRG1 are rather ubiquitously distributed, but in contrast to NDRG1, NDRG3 is predominantly expressed in testis, in the outer layers of seminiferous epithelium, suggesting that it plays a role in spermatogenesis . NDRG4 is quite specific for heart and brain , but its function is unknown. NDRG2 was first cloned in the mouse  and is reportedly expressed essentially in the brain, heart, skeletal muscle and kidney . Recently, we have identified NDRG2 as an early-aldosterone-induced gene in the rat kidney . We have shown by in-situ hybridization that NDRG2 is specifically expressed in the distal parts of the renal tubule and that it is up-regulated rapidly in the rat kidney and distal colon as well as in-vitro in the rat kidney collecting duct RCCD2 cell line . NDRG2 is homologous to MESK2 [misexpression suppressor of dominant-negative KSR (kinase suppressor of Ras)], recently identified in a misexpression screen conducted in Drosophila, suggesting that it may be involved in the Ras signalling pathway . The physiological role of NDRG2 is unknown; some data suggest that it could intervene in epithelial cell differentiation. This gene appears to be expressed abnormally in pancreas and liver cancer . Interestingly, NDRG2 has been identified in transcriptional profiling of the epithelial stem cell niche in skin, as well as in embryonic, neural and haematopoietic stem cells .
Here, we confirm that CCD cells express NDRG2 mRNA and show that AR transcripts, which are highly expressed in PCT cells, are also present in CCD cells. In CCD, the level of expression of AR, measured by real-time PCR, is almost equivalent to that measured in the late proximal tubule (PR). An earlier autoradiographic study mentioned the presence of specific testosterone binding sites in some distal cells (without precise identification) and in inner medullary collecting ducts from rat kidneys . Takeda et al.  have also reported positive AR nuclear staining in the distal nephron, using a polyclonal AR antibody. Together, these results are evidence for the presence of AR in distal or collecting duct cells. The coexpression of NDRG2 and AR in rat kidney CCD raises the possibility of regulation of NDRG2 by androgens. In this in vivo study, we examined the consequences of hormone withdrawal or injection of DHT. Adrenalectomy was necessary, in addition to castration, to reduce both androgens and corticosteroid hormone levels. Castration alone did not modify NDRG2 mRNA in whole kidney extracts, possibly because of unidentified adaptation mechanisms occurring in vivo. Castration led to somewhat elevated (although not significantly) plasma levels of aldosterone, suggesting that castration may trigger complex hormonal compensation(s). Indeed, testosterone inhibits the basal and ANGII- and ACTH-stimulated release of aldosterone production in male rats, via the inhibition of aldosterone synthase activity . Compared with normal rats, castration plus ADX resulted in a significant increase in NDRG2 mRNA expression in whole kidney. A low dose of DHT down-regulated renal NDRG2 significantly due to its effect on the CCD. The mechanisms of the observed reduction in NDRG2 transcript steady-state levels by DHT are unknown but this response may reflect hormonal influence on NDRG2 transcription rate or mRNA stability. The inhibitory action of DHT reported here contrasts with the induction of androgens on the classical androgen-regulated genes (ADH-1, ODC, RP-2, GUS or KAP) in proximal tubule cells [1, 4]. Melià et al.  have also identified novel androgen-regulated genes in the mouse kidney; among them, 16-α-hydroxylase is decreased upon androgen treatment, but its intrarenal expression is not known. Indeed, most, if not all, of the genes up-regulated by androgens appear to be restricted to proximal tubule cells, while NDRG2 represents the first down-regulated gene by DHT in the distal nephron.
The finding that NDRG2 expression is stimulated by aldosterone and repressed by androgens indicate that this gene may be subjected physiologically to a dual opposite steroid hormone control in CCD cells. Future studies should allow evaluating their contribution to NDRG2-mediated functions that remain to be established.
This work was supported by INSERM. S. Boulkroun and C. LeMoellic were recipients of PhD grants of the Ministere de la Recherche et de la Technologie. We are grateful to Dr. A. Vandewalle (INSERM U478, Paris, France) for helpful discussion and critical reading of the manuscript.
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