Kidney International, Vol. 57 (2000), pp. 1295–1298

TRANSGENIC ANIMAL MODELS

Mineralocorticoid receptor knockout mice:Lessons on Na1 metabolism

STEFAN BERGER, MARKUS BLEICH, WOLFGANG SCHMID, RAINER GREGER,and GUNTHER SCHUTZ

Division of Molecular Biology of the Cell I, German Cancer Research Centre, Heidelberg, and the Institute of Physiology,University of Freiburg, Freiburg, Germany

Mineralocorticoid receptor knockout mice: Lessons on Na1 osmolarity and volume, and thereby of blood volumemetabolism. The mineralocorticoid receptor (MR) binds aldo- and blood pressure. Low NaCl concentrations at thesterone and glucocorticoids with equal affinity. In aldosterone macula densa segment of the distal tubule and low bloodtarget tissues, like the epithelial cells of the distal colon and pressure increase renin secretion into the blood andthe principal cells of the collecting ducts in the kidney, the MR

thereby activate the renin-angiotensin-aldosterone sys-is protected from glucocorticoids by the action of the enzymetem (RAAS). Renin cleaves angiotensinogen secreted11b-hydroxysteroid-dehydrogenase type 2 (11bOHSD2), allow-

ing aldosterone to specifically activate the receptor. However, by the liver to angiotensin I. Angiotensin I then is cleavedin MR-expressing cells, which lack 11bOHSD2, like the neu- by the angiotensin-converting enzyme (ACE) to angio-rons of the limbic system in the brain, MR is mainly activated tensin II. By increasing aldosterone secretion and vaso-by glucocorticoids. MR knockout mice die in the second week constriction, angiotensin II causes an increase of bloodafter birth, showing at day 8 symptoms of pseudohypoaldoste-

pressure and thereby an inhibition of the renin releaseronism with hyponatremia, hyperkalemia, high renal salt wast-by the cells of the juxtaglomerular apparatus [6]. Aldo-ing, and a strongly activated renin-angiotensin-aldosterone sys-

tem (RAAS). The activity of the amiloride-sensitive epithelial sterone secretion is dependent not only on angiotensin II.Na1 channel (ENaC) is strongly reduced in colon and kidney, Increased plasma K1 and decreased hepatic aldosteronebut there is no down-regulation of the mRNA abundance of clearance can cause angiotensin-independent rises ofthe three ENaC subunits. Daily subcutaneous injections of plasma aldosterone levels [7, 8].isotonic NaCl solution until weaning and continued oral NaCl

The transepithelial Na1 reabsorption in MR-express-supply lead to survival of the MR knockout mice. The NaCl-ing tight epithelia is mediated by the apical amiloride-rescued MR knockout mice display a strongly enhanced frac-sensitive epithelial sodium channel (ENaC) and the ba-tional renal excretion of Na1, hyperkalemia, and a persistently

strongly activated RAAS. There is almost no renal ENaC activ- solateral (Na1 1 K1)-ATPase. The corresponding genesity. The renal mRNA abundance of aENaC is reduced by 30%, encoding the subunits were long considered as MR targetwhereas bENaC and gENaC are not altered. genes [9]. ENaC is a short-lived protein that is ubiquiti-

nated on the a and g subunits [10]. Nedd4 (neuronalprecursor cell expressed, developmentally down-regu-

The mineralocorticoid receptor (MR), a ligand-acti- lated), a ubiquitin-ligase, was shown to interact with thevated transcription factor, binds with equal affinity aldo- ENaC subunits b and g [11]. It is therefore consideredsterone and glucocorticoids and gets activated by both that ubiquitination plays an important role in the regula-adrenocortical hormones [1]. The MR is expressed in tion of ENaC activity.epithelial cells that line the distal tubules of the kidney, The MR is also expressed in nonepithelial cells, likethe distal colon, and the ducts of salivary and sweat the neurons of the hippocampus [12] and the myocytesglands [2]. In these tight epithelia, aldosterone promotes of the heart [13]. The protection of the nonepithelialMR-dependent sodium reabsorption [3]. Hormone spec- MR from glucocorticoids by 11bOHSD2 is so far notificity in these epithelial cells is achieved by the action established. The nonepithelial MR is therefore to be

considered as a glucocorticoid receptor (GR), as theof the enzyme 11b-hydroxysteroid-dehydrogenase typecirculating plasma levels of glucocorticoids exceed those2 (11bOHSD2), which inactivates glucocorticoids, butof aldosterone by up to three orders of magnitude.not aldosterone [4, 5]. The aldosterone-driven Na1 reab-

sorption is part of the regulation of the extracellular fluidMINERALOCORTICOID RECEPTORKNOCKOUT MICEKey words: aldosterone, receptor, knockout animals, sodium, ami-

loride. To receive new insights into the in vivo function of MRand to separate MR-independent effects of aldosterone 2000 by the International Society of Nephrology

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Berger et al: MR knockout mice and Na1 metabolism1296

Table 1. Phenotypical alterations of mineralocorticoid receptor Table 2. Phenotypical alterations of NaCl-rescued MRknockout miceknockout mice

– Daily injections of isotonic NaCl solution until weaning and contin-– Die between days 8 and 13 after birth– Fractional renal excretion of Na1 is strongly enhanced ued NaCl supply lead to survival

– Fractional renal excretion of Na1 is strongly enhanced– Hyponatremia– Hyperkalemia – Normal plasma Na1

– Hyperkalemia– Plasma levels of renin, angiotensin II, and aldosterone are verystrongly increased – Plasma levels of renin and aldosterone are strongly increased

– ENaC activity in kidney is almost absent– ENaC activity in kidney and colon is strongly reduced but not com-pletely absent – mRNA abundance of aENaC in kidney is reduced by 30%

– mRNA abundance of bENaC, gENaC and Nedd4 in kidney is not– mRNA abundance of ENaC- and (Na1 1 K1)-ATPase subunits inkidney and colon and of Nedd4 in kidney is not altered altered

– Hyperplasia of the extraglomerular mesangium, extension of reninproducing granular cells upstream along the afferent arteriole, hyper-plasia of interlobar arteries

NaCl-RESCUED MINERALOCORTICOIDRECEPTOR KNOCKOUT MICE

action, we established the MR knockout mouse [14]. The Daily subcutaneous injections of isotonic NaCl solu-MR gene was inactivated by homologous recombination tion from day 5 after birth until weaning and continuedin mouse embryonic stem cells. Mice homozygous for oral NaCl supply lead to the survival of MR knockoutthe inactivated MR allele, the MR knockout mice, die mice [16]. This NaCl2 rescue proves that neonatal MRin the second week after birth from dehydration by renal knockout mice die because they are not able to compen-sodium and water loss. In the first week of life, the MR sate for their renal Na1 loss. The injections of isotonicknockout mice develop symptoms of pseudohypoaldo- NaCl solution enable the animals to live through a criticalsteronism. Analysis of eight-day-old MR knockout mice phase of life, after which they adapt their salt and waterrevealed the following alterations (Table 1). They display intake to their persisting renal salt wasting.hyponatremia, hyperkalemia, and a strongly activated The analysis of one-month-old NaCl-rescued MRRAAS, with renin being increased 440-fold, angiotensin II knockout mice revealed the following observations (Ta-being increased 50-fold, and aldosterone being increased ble 2). The adult NaCl-rescued MR knockout mice have65-fold. The ENaC activity of eight-day-old MR knock- a fourfold increased fractional renal excretion of Na1

out mice, measured by the effect of amiloride on the and are hyperkalemic, whereas plasma Na1 is normal.fractional renal excretion of Na1 and on colonic transepi- The RAAS of NaCl-rescued MR knockout mice is stillthelial voltage, was in kidney 24% and in colon 16% strongly activated, with renin and aldosterone being atcompared with wild-type mice. Therefore, ENaC activity least tenfold increased [16]. This indicates that the ani-is strongly reduced but not completely absent. The deter- mals have a Na1 deficit caused by their persisting renalmination of the mRNA abundance of the ENaC- and Na1 wasting. The observed hyperkalemia may also con-(Na1 1 K1)-ATPase subunits in kidney and colon of tribute to the elevated plasma aldosterone level. Amilor-MR knockout mice at day 8 revealed no difference [14]. ide had no effect on the fractional renal excretion ofInvestigation of the mRNA abundance of Nedd4 in the Na1 in NaCl-rescued MR knockout mice, indicating thatkidney also revealed no alteration in MR knockout mice the ENaC activity in the kidney is almost absent [16].compared with wild-type mice. The MR knockout mice The determination of the mRNA abundance of theshow that the regulation of Na1 reabsorption via MR is ENaC subunits and of Nedd4 in kidney of NaCl-rescuednot achieved by transcriptional control of the genes that MR knockout mice revealed a reduction of aENaC byencode the ENaC and (Na1 1 K1)-ATPase subunits, as 30%, but no difference in bENaC, gENaC, and Nedd4.well as Nedd4. Whether this 30% reduction of aENaC mRNA contrib-

The eight-day-old MR knockout mice already showed utes to the almost absent ENaC activity in the kidney ofprominent histological changes at the glomerular vascu- NaCl-rescued MR knockout mice has not been resolved,lar pole. An enlargement of the macula densa segment since there are no data about ENaC protein expressionof the distal tubule, hyperplasia of the extraglomerular presently available.mesangium, extension of renin producing cells upstreamalong the afferent arteriole, and hyperplasia of the in-

MINERALOCORTICOID RECEPTORterlobar arteries were seen [14, 15].KNOCKOUT MICE: A VERSATILE TOOL TOThe MR knockout mice demonstrate the essential roleINVESTIGATE MINERALOCORTICOID ANDof the MR in Na1 homeostasis in a period of life that isGLUCOCORTICOID SIGNALINGcharacterized by a dramatic requirement of Na1 and

The MR knockout mice demonstrate that the MR iswater under the limited NaCl and water supply by moth-er’s milk. not crucial during prenatal development but is important

Berger et al: MR knockout mice and Na1 metabolism 1297

anismen,” the Fonds der Chemischen Industrie, the European Commu-for the adaptation to the limited Na1 supply after birth.nity through grant PL 96,0179, the BMBF through the HGP grant

The survival of the NaCl-treated MR knockout mice 01 KW 9606/7, the Hermann von Helmholtz-Gemeinschaft DeutscherForschungszentren (HGF) through the “Strategiefonds,” and Boeh-allows the investigation of the function of the MR inringer Ingelheim.adult animals and consequently analyses that were thus

far not possible because of the size and age of the neona- Reprint requests to Prof. Dr. Gunther Schutz, German Cancer Re-search Centre, Division Molecular Biology of the Cell I (A0200), Imtal MR knockout mice.Neuenheimer Feld 280, 69120 Heidelberg, Germany.The neurons of the hippocampus are one site of nonep-E-mail: g.schuetz@dkfz-heidelberg.de

ithelial expression of the MR in which the MR is mainlyactivated by glucocorticoids. The hippocampal MR is

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