ARTICLES J Am Soc Ncphrol 9: 1347-1358. 995 1 1 f3-Hydroxysteroid Dehydrogenase, Mineralocorticoid Receptor, and Thiazide-Sensitive Na-Cl Cotransporter Expression by Distal Tubules MAGDALENA BOSTONJOGLO,* W. BRIAN REEVES, ROBERT F. REILLY, HEINO VELAZQUEZ,* NOREEN ROBERTSON,1’ GERALD LITWACK,1’ PETER MORSING,9’ JENS D#{216}RUP,# SEBASTIAN BACHMANN,* and DAVID H. EWSON *D,)pc1,..t,,ze,,t of Anatomy, Charit#{233}, Humboldt U,iit’eis’it’, Berlin, Germans’: Department of Intertial 4’h’diei,u’, Univeisitv ()/AFkallS(1S Sc/uol of Medicine, Little Rock, Arkansas; Depatii:eizt of Internal Medicine, U,zit’e,-sitv of Colo,ado Sc/tool of Medicine (j,7(/ Vete,v,z.v Ad,ninistratioiz Mt’dhal Cente,, De,rier, Colorado; : Yale U,,ii’e,it’ Sc/tool of Medicine and Veterans Ad,ninistration Medical Cetiter, West Haven, Connectictit; ‘Depai-tnzeiit 01 Phari;iacologv. Thomas Jefferson ,7j5r(.#{231}j. Philadelphia, Penn.s’vlt’ania; 1A.s’tra-H#{228}.s-sl#{233}, Moindal, Stt’edeii; (i,il #llls.tittlt(, ()fAFlatoFnv, U,iii’eis’itv ofAarhit.s’, Acirinis, Denmark. Abstract. M ineralocorticoid hormones regulate salt transport polyclonal antibody was generated to localize the thiazide- along the distal nephron by binding to intracellular receptors sensitive Na-Cl cotransporter. Thiazide-sensitive Na-Cl co- and activating gene transcription. Previous experiments transporter and 1 1-hydroxysteroid dehydrogenase expression showed that systemic aldosterone infusions stimulate thiazide- were examined using both in situ hybridization and immuno- sensitive Na and Cl transport by distal convoluted tubule cytochemistry: Na/Ca exchanger and mineralocorticoid recep- (DCT) cells: this ef’fect could have been direct or secondary to tor expression were examined by immunocytochemistry. The systemic hormonal effects. Aldosterone target tissues express results indicate that I I -hydroxysteroid dehydrogenase is cx- both mineralocorticoid receptors and the metabolic enzyme pressed by DCT cells, as well as connecting tubule cells and I I f3-hydroxysteroid dehydrogenase type 2. Mineralocorticoid principal cells of the collecting duct: expression levels are low receptors have been localized to the DCT in some experiments. near the junction with the thick ascending limb and rise near but not in others. Expression of I I -hydroxysteroid dehydro- the transition to the connecting tubule. Mineralocorticoid re- genase type 2 by DCT cells has not been investigated. The ceptors are expressed by DCT cells, as well as along the thick present experiments were designed to test the hypothesis that ascending limb, connecting tubule, and collecting duct. The rat DCT cells are targets of aldosterone action. Patterns of results indicate that components of the mineralocorticoid re- mineralocorticoid receptor. I I -hydroxysteroid dehydroge- ceptor system are expressed by DCT cells, suggesting that nase, thiazide-sensitive Na-Cl cotransporter. and Na/Ca ex- these cells are targets of aldosterone action. changer expression along the distal tubule were examined. A Mineralocorticoid hormones contribute importantly to the so- controversial. Recently, aldosterone was shown to stimulate diui’n retention that occurs during extracellular fluid volume thiazide-sensitive Na-Cl cotransport by renal distal tubules. in contraction. Principal cells of the cortical collecting duct are fritO. Aldosterone infusion into adrenalectomized rats in- important targets of aldosterone action ; aldosterone increases creased the thiazide-sensitive component of sodium transport the number of amiloride-sensitive Na channels at their apical by perfused distal tubules up to 20-fold (2). This change in membranes and the number of ouabain-sensitive Na/K ATPase functional capacity was accompanied by large increases in the pumps at their basolateral cell membranes (I ). Other nephron number of high-affinity receptors for the thiazide-like drug segments may respond to aldosterone. but the data are more I3Hlmetolazone (2,3). Because thiazide-sensitive Na-Cl co- transporters are expressed primarily by distal convoluted tu- bule (DCT) cells (4-6), the functional data raised the possi- Received October 5. 1997. Accepted February 24. 1998. bility that DCT cells are targets of aldosterone action. Correspondence to Dr. 1)avid H. Ellison. Section of Nephrology/I 1 IC. Denver Alternatively, the effects of systemic aldosteronc infusion VA Medical Center. 055 Clermont Street. Denver. CO 80220. could have been secondary to systemic actions of the hormone I 046-6673/0908- I 347$03.O0/0 Journal of the American Society of Nephrology or effects of aldosterone on nephron segments that do not Copyright 0 998 by the American Society of Nephrology express the thiazide-sensitive Na-Cl cotransport. I 348 Journal of the American Society of Nephrology J Am Soc Nephrol 9: 347-1358, 1998 Aldosterone acts by binding to intracellular mineralocorti- the distal nephron suggests functional heterogeneity in aldo- coid receptors, which then bind to hormone response elements sterone sensitivity. in the promoters of genes (7). Mineralocorticoid receptors, however, are not specific for mineralocorticoids, but also bind Materials and Methods endogenous glucocorticoids, such as cortisol and corticoste- Antibodies rone, with nearly equal affinity (8). In aldosterone target cells, The amino-terminal putative cytoplasmic domain of the mouse glucocorticoids do not bind to mineralocorticoid receptors be- thiazide-sensitive Na-Cl cotransporter (mNCC, GenBank accession cause the enzyme I I -hydroxysteroid dehydrogenase type 2 no. U6l085. amino acids I through 98) ( 17) was amplified by PCR rapidly metabolizes glucocorticoid hormones (9). An inherited and cloned into pBluescript. The product was sequenced, excised deficiency of this enzyme leads to the syndrome of apparent using EcoRI and Hindlll, and cloned into the maltose-binding protein expression vector pMAL (New England BioLabs. Beverly, MA). The mineralocorticoid excess because glucocorticoid hormones are construct was verified by sequencing. and production of the mNCC- not metabolized, bind to rnineralocorticoid receptors in aldo- maltose binding protein fusion was induced by incubating trans- sterone target cells, and stimulate sodium reabsorption consti- formed Eseheriehia -o/i with 0.3 mM isopropyl b-o-thiogalactopyr- tutively (10). anoside for 2 h. The cells were then lysed. and the fusion protein was Sites of mineralocorticoid receptor expression and purified over an amylose affinity column as described (18). New I IHSD2 expression along the distal nephron have been Zealand White rabbits were immunized with 100 g of fusion protein investigated previously. using a variety of techniques. Both in coniplete Freund’s adjuvant and boosted with 50 j.tg of fusion the receptors and the enzyme have been shown to be cx- protein in incomplete Freund’s adjuvant every 6 wk. pressed by cortical collecting tubule cells, but evidence of The polyclonal rabbit antibodies to human I I HSD2 and to the Na/Ca exchanger were described and characterized previously expression by other nephron segments has been more con- ( 14,19). The anti-Tamm-l-lorsfall antibody was kindly provided by troversial. l3HlAldosterone bound to isolated rabbit kidney John Hoyer (University of Pennsylvania. Philadelphia. PA) (20). The tubules throughout the collecting duct, but not along the anti iiii neralocorticoid antibody was generated agai nst a synthetic pep- DCT ( I I ): in contrast, autoradiographic studies, using tide and has been characterized previously (21). I3Hjaldosterone, showed specific labeling not only of col- lecting ducts, but also of DCT (12). Labeling of DCT (and Western Blotting of tiiedullary collecting tubules). however, required higher Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was concentrations of radioligand than did labeling of cortical carried out using the discontinuous buffer system of Laemmli (22). collecting tubules. This prompted Marver to suggest that Proteins were separated on 10% (mouse) and 6% (rat) polyacrylamide two classes of aldosterone receptor are present along the gels and transferred to polyvinylidene difluoride paper (Imobilon-P. DCT ( 12). Recently. I I USD2 was shown to be expressed by Millipore. Bedford. MA). The blot was rinsed two to three times with “distal tubules” and collecting ducts in rat and human kid- phosphate-buffered saline (PBS)-Tween 20. incubated with Blotto-T ney, but whether expression extended into the DCT was not (5% nonfat dry milk. 0. 1 % Tween 20 in PBS), and then with the examined specifically (13-16). primary antibody. diluted 1:1(X). The blot was washed and then incubated with horseradish peroxidase-coupled goat anti-rabbit The present studies were designed to determine where along (Zymed Laboratories, South San Francisco. CA). After washing. the the distal nephron high-level expression of I I HSD2 and mm- strips were developed using the Pierce Supersignal Substrate Western eralocorticoid receptors begins and. more specifically, to test blotting system (Pierce. Rockford. IL). the hypothesis that DCT cells are targets of mineralocorticoid action. To test this hypothesis. combined in situ hybridization Rat Kidney Fixation and immunocytochemical approaches were used to identify Five male Sprague Dawley rats (300 to 50() g body wt) were expression sites along the rat distal nephron. The results mdi- allowed free access to standard laboratory diet and tap water. For cate that I I HSD2 and mineralocorticoid receptors are cx- perfusion fixation. animals were anesthetized