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Home , Aldosterone, Mineralocorticoid

ARTICLES J Am Soc Ncphrol 9: 1347-1358. 995

1 1 f3-Hydroxysteroid Dehydrogenase, , and -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 regulate salt transport polyclonal antibody was generated to localize the thiazide- along the distal by binding to intracellular receptors sensitive Na-Cl cotransporter. Thiazide-sensitive Na-Cl co- and activating . Previous experiments transporter and 1 1-hydroxysteroid dehydrogenase expression showed that systemic infusions stimulate thiazide- were examined using both in situ hybridization and immuno- sensitive Na and Cl transport by 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 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 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 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 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 (7). Mineralocorticoid receptors, however, are not specific for , but also bind Materials and Methods endogenous , such as 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 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 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 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 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 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 . For cate that I I HSD2 and mineralocorticoid receptors are cx- perfusion fixation. animals were anesthetized (pentobarbital. 40 pressed by the DCT, but variation in relative expression along mg/kg body wt. intraperitoneally) and kidneys were fixed by retro- grade perfusion through the abdominal aorta with freshly prepared 3% paratormaldehyde. buffered in PBS at pH 7.4 fcr 5 mm as described (23). To protect the tissues from freezing artifacts, kidneys were .i This entyme. I I -hydroxysteroid dehydrogenase type 2. is related to hut subsequently soaked in a sucrose-PBS solution adjusted to 800 distinct from I I -hydroxysteroid dehydrogenase type I . an enzyme expressed mosmol/kg. Kidneys were then removed and cut into slices of ap- in the and the renal proxinial tubule. The type 2 enzyme is dependent on proxii’nately 3 mi’n thickness. The slices were shock-frozen in liquid NAD (rather than NA[)P), has a higher affinity for (and corti- nitrogen-cooled isopentane. sol). and is expressed by aldosterone target tissues ( I 5. I6). In this article. the abbreviation I I HSD2 will he used to designate this enzyme. i, The nephron segment between the region of the macula densa and the Preparation of Riboprohes confluence with another tubule has been called the “distal tuhule’ and the A rihoprohe for the thiazide-sensitive Na-Cl cotransporter was distal convoluted tubule.’ According to nomenclature recommended by the prepared from a 7l3-bp partial eDNA fragment of mNCC (24), Renal Commission of the International Union of Physiological Sciences (42). this nephron segment comprises three major subsegments. the distal convo- comprising putative membrane-spanning domains I through 7. and luted tubule. the ci.)nnecting tubule. and the initial portion of the cortical has been described previously (5). The I IHSD2 probe was generated collecting tubule. In this article. the terni distal convoluted tubule is used to by PCR from rat kidney eDNA ( 16). The primers were +389 (5’- designate the true or early’ distal convoluted tubule. CCGGTTGTGACACTGGTTTT-3) and -637 (5’-TGAGGCCAG- J Am Soc Nephrol 9: I 347- 1358. I998 Cotransporter Expression by l)istal Tubules I 349

CATTGTTAACC-3’ ): they generated a 248-nucleotide fragment that was cloned into the EcoRV site of pBluescript KS+ and sequenced. RNA probes were synthesized and labeled by in t’ilio transcription using digoxigenin-l l-UTP (25). For the NCC. an antisense probe was prepared by linearizing using Xhol and transcribed using T7 RNA polymerase. To obtain the probe (control). plasmid was linear- ized using Xbal and then transcribed using T3 RNA polymerase. For 1 1USD2. an antisense probe was generated by linearizing with Hin- dill and transcribed with T7 RNA polymerase. The sense probe was prepared by linearizing with BwnHl and transcribing with T3 poly- merase. Because RNA probes for in situ hybridization should not exceed the length of 500 bases. the probes were shortened by alkaline hydrolysis to an average length of 180 to 200 bases. 220- in Situ Hvbridizatioiz iiid lmnmnunocvtocheimzistrv Cryostat sections (5 to 7 p.m thick) were thawed onto silanized glass slides. For in situ hybridization. slides were processed essen- tially as described (25). The optimal concentrations of the digoxigenin (DIG)-laheled antisense probe was between 5 and It) ng/pi hybrid- ization mixture. Hybridization was perfornied at 40#{176}Cfor 16 to 18 h. Slides were washed at 40#{176}Cfor 30 mm in 2x SSC (SSC: 150 mM -* NaCI, 15 mM NaCitrate). followed by two washes for 30 mm in 0.375X SSC containing 50% formamide and another wash for 30 mm in 0. 1 X SSC containing 50)c Iormaiiiide. Subsequently. slides were 46- rinsed twice in 0.SX SSC at room temperature for 10 mm followed by a rinse in 0.2N SSC for 10 mm followed by two rinses in buffer I ( lOt) mM Tris-HCI. 150 mM NaCl. pH 7.5). Blocking of nonspecific ...1 antibody binding sites was then performed by incubating sections with modified blocking medium ( Boehringer Mannheim). Mouse Kidney Rat To identify sites of gene expression. combined in situ hybridization Kidney and immunohistochemistry was performed on the same tissue section using different probes. After removal of’ the blocking medium, a Fi,i’ure I. Immunoblot of rat and mouse kidney cortex. Mouse kidney mixture of’ sheep anti-DIG-alkaline phosphatase conjugate and one of’ cortical membranes (left) blotted with Na-Cl cotransporter (NCC)- the specil’ic antibodies were diluted in blocking medium (dilution of NAbl. Preimmune serum did not recognize membrane proteins. anti-DIG-conjugate was I :500. dilutions of the other antibodies were whereas NCC-NAbl recognized a broad band between 140 and 190 I :30 for anti-Tamm-Horsfall protein; I : I 00 for anti-NCC. anti- kD. Blocking with maltose-binding protein (MBP-Blocked) had no 1 1 HSD2. and anti-mineralocorticoid receptor: and I :20 for anti-Na I effect. but blocking with the fusion protein (FB-Blocked) completely Ca2 exchanger. This mixture was applied to the sections for 2 h at blocked binding. Rat kidney membranes were probed with NCC- rOOill teniperature in a moist chamber. followed by 16 h of incubation NAbI (right). The antibody recognized a protein hand in the range of at 4#{176}C.The slides were then washed twice. and antibodies were 140 to 190 kD in rat kidney cortex; in addition. a variable band. near detected using indirect immunotluorescence (25). Message was de- 47 kD. was observed on sonic blots; this hand was blocked by tected using the nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl- incubating with the fusion protein and may represent a proteolytic phosphate systeni as described (25). fragment or the alternatively spliced isolorm identified by Hoover ci The specificity of the immunohistocheniical staining in the present a!. (54). experiments was tested by replacing the respective antibody with nonimmune seruni or I X PBS; in all cases. no signal was obtained under this condition. The specificity of the in situ hybridization signal was verified by parallel incubation with sense and antisense ribo- preincubation of the serum with maltose-binding protein alone probes. Throughout all of the experiments, the sense probes did not had no effect. Plotkin ci al. (4) reported that the broad hand produce any detectable signal. recognized by an anti-NCC antibody could he resolved into several discrete bands. In the present experiments, the broad Results band recognized by mNCC-NAbI was diffuse on a 6% sodium Figure 1 shows an immunoblot using antibody mNCC- dodecyl sulfate gel: although discrete hands within this region NAbI. The results indicate that the antibody recognizes a were not detected, a suggestion of sub-bands is apparent. protein band in the range of 140 to 190 kD in both rat and Figure 2 compares expression of the thiazide-sensitive mouse kidney cortical tissue. Antibody specificity was docu- Na-Cl cotransporter in rat kidney cortex as detected by in situ mented by showing that preimmune serum does not react hybridization, using an mNCC-specifIc rihoprohe, and immu- against mouse (or rat. data not shown) kidney tissue and by nocytochemistry, using antibody mNCC-NAhl. on the same showing that preincubation of the serum with an excess of the kidney section. This low-power view reveals nearly complete fusion protein blocked binding to the 140- to 190-kD band; overlap of expression. as detected using either technique. in- I 350 Journal of the Anserican Society of Nephrology J Am Soc Nephrol 9: l347-l358. 1998

Fi,#{231}ii.e2. Localization of thiazide-sensitive Na-Cl cotransporter (NCC) in rat kidney cortex. A and C show in situ hybridization for NCC using an antisense digoxigenin-laheled rihoprobe. B and D show the same fields stained with the NCC-NAh I . Note the extensive and nearly complete colocalization of NCC expression detected using the two techfliques and the transition from distal convoluted tubules (DCT) to connecting tubules (CNT). indicated by the lines in C and D.

Magnification: X I6() in A and B; X226 in C and D.

dicating that sites of NCC expression can he detected using either technique.

Figure 3 shows the pattern of I 1 USD2 expression in rat I’I/1II-c 3. Overview of I l/3-hydroxysteroid dehydrogenase type 2-posi- kidney cortex, as detected by in situ hybridization and immu- tive tubular portions in rat kidney corteX and medulla as revealed by in nocytochemistry on similar kidney sections. These low-power silil hybridization with antisense digoxigenin-labeled I I -hydroxysteroid views indicate that expression is limited to a minority of tubule dehydrogenase type 2 ribeiprobe (A) and immunocytochemistry using Texas red second antibody detection (B and C). Analogous portions of profiles throughout the kidney cortex. when detected by either the cortex have been selected in A and B. I I -Hydroxysteroid dehydro- ill .s’iti hybridization (Figure 3A) or immunocytochemistry genase type 2 expression is limited to a minority of’ tubule profiles in A (Figure 3B). I IHSD2 expression extends into the medulla and B. Panel C shows the . Strong labeling fbr I I - (Figure 3C), but the intensity of staining declined from the hydroxysteroid dehydrogenase type 2 is found in the outer stripe of the cortical to the medullary collecting tubule (similar results were outer medullary collecting duct (top half of the micrograph). whereas in observed by iii situ hybridization but are not shown). Simul- inner stripe of the outer medullary collecting duct. the signal intensity is taneous iii situ hybridization and immunocytochemistry on the weaker. In the inner medullary collecting duct. profiles are mostly neg- same kidney section using I I HSD2-specific probes indicated ative. Magnification. X50. J Aim Soc Nephrol 9: 1347-1358. 1998 (‘otransporter Expression by l)istal Tubules I 35 I

that in situ hybridization and imniunocytochemistry for profiles do not react with either probe and are proximal tu- I I HSD2 identify the same tubule population (data not shown). bules. Some tubule profiles express the thiazide-sensitive Previous results from human kidney indicated that I I HSD2 Na-Cl cotransporter but do not express I I HSD2 (Figure 6, is expressed at low levels along the thick ascending limb. To arrows). Other tubule profiles express I I HSD2 hut do not investigate whether 1 IHSD2 expression extends proximally express the thiazide-sensitive Na-Cl cotransporter (Figure 6, into the thick ascending limb of rat kidney. I I HSD2 expres- arrowheads). A third group of tubule profiles expresses both sion in kidney cortex was compared with Tamm-I-Iorsfall the thiazide-sensitive Na-Cl cotransporter and I I HSD2 (Figure protein expression. Tamm-Horsfall protein specifically labels 6, asterisks). Although expression ot 1 1 USD2 appears to be thick ascending limb cells in the rat (26). Figure 4 shows that less intense along segments that coexpress the thiazide-sensi- 1 1 HSD2 expression is not detected in Tamm-Horsfall-positive tive Na-Cl cotransporter, this difTerence may result from thick ascending limb cells. quenching of the I I HSD2 signal by the in situ hybridization Figures 5 and 6 compare sites of NCC and I I HSD2 expres- reaction (see below). sion along the rat distal nephron. Figure 5 shows sites of NCC We have previously suggested that the rat DCT comprises gene expression. as detected by immunocytochemistry. and two subsegments. the DCTI and DCT2 (see Discussion and sites of I I HSD2, as detected by in situ hybridization, on the reference 5). To deterniine whether the onset of I IHSD2 same kidney sections. One population of tubule profiles cx- expression corresponds to the junction between DCTI and presses NCC but not I I HSD2 (Figure 5, arrows). A second DCT2, sites of Na/Ca and I I HSD2 expression were compared. population of tubule profiles expresses I I HSD2 but not NCC Figure 7. A and B. shows that most tubule profiles that express (Figure 5, asterisks). A third group of tubule profiles expresses I I USD2 in the superficial kidney cortex also express the both 1 1HSD2 and the NCC (Figure 5. + symbol). Figure 6 Na/Ca exchanger: a transition from an I I HSD2-negative seg- shows a higher power view of NCC and I I HSD2 expression in ment tO an I I HSD2-positive segment is indicated by the line. rat kidney cortex, as detected using in situ hybridization ftr An identical site of transition from Na/Ca-negative to Na/Ca- NCC and immunocytocheniistry for I I HSD2. Most tubule positive is seen in the same tubule profile. Note that although

Figure 4.Localizatioii of’ 1 1 -hydroxysteroid dehydrogenase in rat kidney cortex as revealed by combined application of in situ hybridization for I 1f3-hydroxysteroid dehydrogenase (A) and inimunohistocheniistry with antibody against Tanim-Horsfall protein (B). (A) I 1/3-Hydrox- ysteroid dehydrogenase can be localized in profiles of the CNT. including an arcade (arrows) next to an interlohular artery. whereas the thick ascending limb (TAL) (shown in B with TAL cells positive and macula densa cells unreactive) is unreactive. Magnilication. X 140. I 352 Journal of the Anierican Society of Nephrology J Ani Soc Nephrol 9: 1347-1358. 1998

mineralocorticoid receptors are expressed by Na/Ca exchang- er-negative (DCT1 ) and Na/Ca exchanger-positive (DCT2) portions of the DCT. Figure 1 1 shows that mineralocorticoid expression is detected in connecting tubule cells together with the Na/Ca exchanger, as well as beyond the distal extent of Na/Ca exchanger expression, along the cortical collecting duct. Mineralocorticoid receptors are not detectably expressed by a minority cell population in the connecting tubule and collecting duct, a cell population that does not express the Na/Ca cx- changer either. This characteristic identifies these cells as intercalated cells.

Discussion Mineralocorticoids are potent sodium-retaining that participate importantly in extracellular fluid volume homeosta- sis and BP control. The mineralocorticoid hormone aldosterone increases sodium reabsorption, at least in part, by binding to mineralocorticoid receptors inside cells and activating gene

transcription ( I ). Mineralocorticoid target cells have been iden- tified using functional techniques and by examining sites of mineralocorticoid receptor expression. The cortical collecting duct, where aldosterone stimulates electrogenic sodium trans- port, is a well established target of mineralocorticoid action, but other nephron segments may respond to aldosterone as well. Adrenalectomy reduces Na/K ATPase activity along the thick ascending limb and distal tubule. as well as the cortical collecting duct (27-29). Aldosterone infusion into adrenalec- tomized animals stimulates sodium absorption by loops of Henle (30) and distal tubules (2), and stimulates proton secre- tion by medullary collecting ducts (3 1). In the present studies, mineralocorticoid receptor expression was detected throughout the distal nephron, beginning in the cortical thick ascending limb. Expression was detected in DCT, Ei,tiii’e 5. Localization of I I /3-hydroxysteroid dehydrogenase in rat kidney cortex as revealed by conihined application of in situ hybrid- connecting tubules, and cortical collecting ducts. In the con- ization for I I /3-hydroxysteroid dehydrogenase (A and C) and immu- necting tubule, expression by intercalated cells was not de- nohistochemistry with antibody against NCC ( B and D). NCC can be tected. The present results generally agree with previous stud- localized in profiles of the DCT I (arrows in B). whereas I 1/3- ies, in which immunocytochemical techniques were used. Sites hydroxysteroid dehydrogenase is negative (arrows in A). In DCT 2. of mineralocorticoid receptor expression along the nephron ( + signs in A and D). both NCC and 1 1/3-hydroxysteroid dehydro- have been examined previously by nephron segment PCR (32), genase are expressed. In CNT (asterisks in A through D). 1 1/3- by radioligand binding ( 1 1 ) and autoradiographic ( I 2) tech- hydroxysteroid dehydrogenase. hut not NCC. is expressed. Magnifi- niques. and by immunocytochemistry (2 1 .33-36). All tech- cation. X420. niques have shown high-level mineralocorticoid receptor cx- pression by cortical and medullary collecting tubules, but expression along other segments has been less consistently immunocytochemical methods are not quantitative. expression detected. Immunocytochemical techniques detected mineralo- levels of I I HSD2 at the transition point appear quite high, corticoid receptor expression along the DCT (33-36), whereas providing evidence that the lower level expression of I I HSD2 I3Hlaldosterone binding studies did not ( I I ). Autoradiographic along segments that express NCC. observed in Figure 7, is in studies using 13H]aldosterone did detect specific labeling along part due to quenching. Figure 7, C and D, indicates that some the DCT (and the cortical thick ascending limb), but binding to linear tubule profiles within medullary rays express I I HSD2 these sites required 5- to 10-fold higher I3Hlaldosterone con- but not the Na/Ca exchanger. centrations than required for binding to cortical collecting Localization 01 the niineralocorticoid receptor in the region tubules ( I 2). On the basis of these results. Marver concluded of the distal nephron is shown in Figures through 1 1 . Figure that DCT express two different classes of mineralocorticoid 8 shows that a distinct signal was detected along the cortical receptor (12). thick ascending limb by the antimincralocorticoid receptor During the past several years, the role of the enzyme antibody. Figure 9 shows that the signal was frequently weaker I 1HSD2 in maintaining mineralocorticoid hormone specificity and more variable in macula densa cells. Figure 10 shows that has been elucidated, and the enzyme has been expressed and J Am Soc Nephrol 9: 1347-1358. 1998 Cotransporter Expression by Distal Tubules 1353

Figure 6. Localization of 1 1f3-hydroxysteroid dehydrogenase in rat kidney cortex as revealed by combined application of in situ hybridization for the thiazide-sensitive Na-Cl cotransporter (A) and immunohistochemistry with the antibody against 1 lf3-hydroxysteroid dehydrogenase (B). NCC can be localized in profiles of the DCT including DCI I (arrows) and DCT 2 (with weaker label, asterisks). Connecting and collecting ducts (arrowheads, A and B) express I 1/3-hydroxysteroid dehydrogenase. but do not express the thiazide-sensitive Na-Cl cotransporter. Immunoreactivity for 1 1 /3-hydroxysteroid dehydrogenase is weak in portions of DCT 2 in part because fluorescence is reduced due to underlying in situ hybridization signal for NCC. Magnification: X21 1 in A and B.

cloned (13,37). As expected, based on its postulated physio- The present experiments were designed to examine miner- logic role, 1 1 HSD2 was shown to be expressed by nephron alocorticoid receptor and I I HSD2 expression along the rat segments that express mineralocorticoid receptors. Roland et distal nephron. To identify DCT cells and permit coexpression al. (38) used radioactive in situ hybridization to show that studies, a polyclonal antibody against the amino terminus of

1 1HSD2 is expressed by cortical distal tubules and medullary the NCC was developed. The antibody, mNCC-NAbl , recog- straight tubules in rat kidney. Smith et a!. (15) raised an nizes a 140- to l70-kD protein in rat and mouse kidney cortex; antibody to the carboxy-terminal region of rat 1 1HSD2. These Plotkin et al. (4) reported a similar result using an antibody investigators showed high-level expression of 1 1HSD2 in rat generated against the amino terminus of the rat NCC. The cortical distal tubules and lower-level expression in tubules specificity of the antibody for NCC was confirmed by showing that were believed to be thick ascending limbs. In these studies, that the maltose-binding protein-fusion protein, but not mal- precise identification of nephron segments was not possible tose-binding protein itself, blocked the signal on Western blot. because colocalization with nephron segment-specific markers mNCC-NAbl labels the apical membrane ofdistal tubule cells, was not used; it appears that profiles identified as “distal in a distribution that is very similar to that demonstrated using tubules” included DCT, connecting tubules, and cortical cot- in situ hybridization in the present experiments (Figure 2). This lecting ducts. Kyossev et al. (14) generated a polyclonal anti- distribution is similar to that reported previously by us, using in body to a fragment of human 1 1HSD2. In human kidney, this situ hybridization and nephron segment PCR (5,6,39), by He- antibody labeled cells of the connecting tubule and collecting bert et a!. using in situ hybridization and immunocytochemis- duct, as identified by colocalization with band 3-positive in- try (4,40), and by Yang et al. (41) using nephron segment PCR. tercalated cells. Low-level staining was also observed along Recent molecular studies have revealed previously unrecog- the thick ascending limb, as identified by colocalization with nized heterogeneity in transport protein expression along the an antibody to Tamm-Horsfall protein. All of these studies rat distal nephron. Morphologic studies indicate that the rat indicate that high-level 1 1HSD2 expression begins along the distal tubule comprises the DCT, the connecting tubule, and an distal tubule, but they do not identify the nephron subsegment initial segment of cortical collecting tubule (42). Gene expres- in which expression begins. sion studies have identified a nephron segment that begins just I 354 Journal of the American Society of Nephrology J Am Soc Nephrol 9: 1347-1358, 1998

Figure 7. Localization of 1 1/3-hydroxysteroid dehydrogenase in rat kidney cortex as revealed by combined application of immunohistochem- istry for I I f3-hydroxysteroid dehydrogenase (A and C) and Na/Ca exchanger (B and D). A and B show a complete overlap of signals in DCT and CNT (transition between [negative] DCT 1 and [positive] DCT 2 is indicated by white lines in A and B). C and D show a region of coexpression, CNT, and the transition (white line) to cortical collecting duct (CCD), where Na/Ca is not expressed. Magnification: X 197 in AandB; Xl80inCandD.

Figure 9. Localization of mineralocorticoid receptor in rat kidney cortex as revealed by immunohistochemistry for mineralocorticoid Figure 8. Localization of mineralocorticoid receptor in rat kidney receptors (A) and corresponding interference contrast microscopy (B). cortex as revealed by immunohistochemistry for the mineralocorticoid Mineralocorticoid receptors are absent from some of the cells of the receptor (A) and corresponding interference contrast microscopy (B). macula densa (arrows). In an adjacent CNT (asterisks), mineralocor- The mineralocorticoid receptor shows a distinct signal in the cortical ticoid receptors show a distinct signal, part of which is located in the thick ascending limb. Magnification: X 540. adluminal compartment.

distal to the thick ascending limb and expresses the NCC but brane, but not the NCC (5,43,44); based on its morphology and not the Na/Ca exchanger (5); based on its location and mor- location, this segment has been identified as connecting tubule. phology, this segment has been identified as the DCT. A A third segment, distal to the connecting tubule, is the initial different segment has been identified in close apposition to portion of the cortical collecting tubule. Principal cells in this intralobular arteries and in arcades; cells in this segment cx- segment express the epithelial Na channel but not the Na/Ca press the Na/Ca exchanger at their basolateral , exchanger or the NCC (5,43). A distal segment is also consis- the epithelial Na channel and aquaporin 2 at their apical mem- tently identified that is distinct from those described above. J Am Soc Nephrol 9: 1347-1358. 1998 Cotransporter Expression by Distal Tubules

the connecting tubule of the rat. In those studies, the connect- ing tubule was identified by the appearance of intercalated cells and expression of the Na/Ca exchanger. When viewed in the context of more recent studies, it is clear that the segment identified as connecting tubule by Plotkin et al. has character- istics consistent with the DCT2. First, it is well established that intercalated cells are present in the rat DCT (42). Furthermore, as we (5) and others (43) have shown, Na/Ca expression begins in the DCT of the rat. Thus, the current results are completely compatible with those reported by Plotkin et al. The present results indicate that rat DCT cells express I IHSD2. Positive identification of DCT cells was made by colocalization with NCC and with the Na/Ca exchanger. Al- though 1 1HSD2 expression levels in DCT cells may be lower than in connecting and collecting tubule cells (Figures 5 and 6), several lines of evidence suggest that they are physiologically significant. First, 1 1HSD2 expression levels at the onset of Figure 10. Localization of mineralocorticoid receptor in rat kidney Na/Ca exchanger expression appear to be nearly as high as cortex as revealed by combined application of immunohistochemistry observed along the collecting duct (Figure 7). We have shown for the mineralocorticoid receptor (A) and the Na/Ca exchanger (B). The mineralocorticoid receptor is positive in the DCT1 (above the previously that Na/Ca exchanger expression begins within the lines) and in the DCT2 (below the lines). as identified by the double DCT in the rat (5). Second, we have shown recently that DCI labeling with the Na/Ca exchanger antibody. segments that express the thiazide-sensitive Na-Cl cotrans- porter actively metabolize corticosterone via 1 1HSD2 when dissected from rabbit kidney (39). Although the present results suggest that DCI cells do express physiologically significant amounts of 1 1HSD2, expression does appear to vary along the length of the distal nephron, increasing from undetectable levels near the junction with the thick ascending limb to relatively high levels near the junction with the connecting tubule. In the present studies, 1 1HSD2 expression was not detected along the thick ascending limb, but several other studies have reported that thick ascending limb cells do express

1 IHSD2 at low but consistent levels (14,15). It is likely that the absence of detectable expression in the present studies resulted from conditions of lower sensitivity. Expression along the medullary collecting tubule was clearly detectable in the present studies, but levels appear lower than along the con- necting tubule and cortical collecting duct. Although the techniques used in the current experiments are not quantitative, the differences in 1 1HSD2 and mineralocor- ticoid receptor expression along the distal nephron may be Figure 1 1. Localization of mineralocorticoid receptors in rat kidney physiologically important. As discussed above, autoradio- cortex as revealed by combined application of immunohistochemistry for mineralocorticoid receptors (A) and Na/Ca exchanger (B). Inter- graphic analysis of [3H]aldosterone binding showed that DCI calated cells (arrows) in the connecting tubule are negative for mm- do bind to aldosterone, but only at higher concentrations ( 12). eralocorticoid receptors and Na/Ca exchanger, whereas connecting The difference between binding to DCI and connecting and tubule cells show a distinct signal fr both (arrowheads). collecting tubules might reflect differences in the relative cx- pression of 1 1 HSD2 and mineralocorticoid receptors. Where cellular concentrations of 1 1HSD2 are insufficient to metabo-

This segment expresses the NCC, the Na/Ca exchanger, and lize glucocorticoids completely (e.g. , medullary collecting duct calbindin 28D (5,45). but does not express the epithelial Na and DCI), mineralocorticoid receptors may be partially occu- channel or aquaporin 2 (43). Based on the unique expression pied by glucocorticoids. Aldosterone may compete for binding, pattern of this segment and its morphologic resemblance to but only at higher concentrations. Such an effect might be DCT, we have referred to it as the DCT2 (5). Ofnote, although variable if the activity of 1 1HSD2 is regulated physiologically. this segment is relatively short in the rat. the DCT2 may be the Nolan et al. (46) showed that I I HSD2 activity is inhibited by predominant segment between the macula densa and the junc- acid pH in inner medullary collecting tubule cells. Thus, in the tion to form the collecting duct in humans (5,42). medulla, low-level 1 1HSD2 expression may “protect” inner Plotkin ci al. (4) reported that NCC expression extends into medullary collecting duct cells from glucocorticoids when the I 356 Journal of the American Society of Nephrology J Am Soc Nephrol 9: 1347-1358, 1998

Table 1. Expression of ion transport proteins, metabolic enzymes, and hormone receptors along the distal nephron, as revealed by the current experimentsa

Parameter TAL DCT1 DCT2 CNT CCT

NCC 0 ++++ +++ 0 0 Na/Ca 0 0 +++ ++++ 0

11HSD2 0(+/-) 0 ++ ++++ +++ MR + + ++ ++ ++ AQP-2 0 0 0 +++ +++

a Of note, the techniques used were semiquantitative. and the scheme 0-+ + + + is used to indicate relative expression level as detected by eye. Expression of I I /3HSD was not detected along the thick ascending limb in these experiments, but previous work using more sensitive techniques has detected low level expression along the thick ascending limb in rat (14). Data on AQP-2 expression are from references 43 and 53. NCC, thiazide-sensitive Na-Cl cotransporter; Na/Ca, Na/Ca exchanger; 1 1HSD2, 1 1/3-hydroxysteroid dehydrogenase; MR. mineralocorticoid receptor; AQP-2, aquaporin-2; TAL, thick ascending limb; DCT1 and DCT2, Na/Ca exchanger-negative and Na/Ca exchanger-positive portions of the DCT, respectively; CNT, connecting tubule cells; CCI, cortical collecting tubules.

pH is high, but permit glucocorticoid occupation of ascending limb to the medullary collecting duct. DCT express mineralocorticoid receptors when urine pH is low. both mineralocorticoid receptors and I I HSD2, together with The present results show that DCT cells are targets of the thiazide-sensitive Na-Cl cotransporter. This result provides aldosterone action. The cells express both mineralocorticoid a molecular basis for the observation that aldosterone regulates receptors and the enzyme 1 1HSD2, as well as the thiazide- thiazide-sensitive Na-Cl cotransport in vivo; the result identi- sensitive Na-Cl cotransporter. Aldosterone was shown previ- fies DCI cells as targets of aldosterone action. ously to stimulate Na and Cl transport rates by up to 20-fold, compared with rates in adrenalectomized animals. Although the current data do not establish that the effects of aldosterone Acknowledgments This work was supported by a Grant-in-Aid from the American on thiazide-sensitive Na and Cl transport are direct (i.e. , slim- Association, by Research Grant 1 P50 HL55007 from National ulate the transporter directly), they do suggest that systemic Institutes of Health, by the Department of Veterans Affairs, and by the effects of aldosterone may not be required to upregulate thia- Deutsche Forschungsgemeinshaft (Ba 700/10-1). It was also sup- zide-sensitive Na-Cl cotransporter activity. This would be ported by the Forschungsforderungsfond of the Charit#{233},Berlin, and by compatible with the observation that both dietary NaC1 restric- Research Grant DK 44441 from National Institutes of Health (to Dr. tion (with extracellular fluid volume contraction; reference 47) Litwack). This research was conducted during the tenure of an Es- and chronic aldosterone infusion (with extracellular fluid vol- tablished Investigatorship of the American Heart Association (Dr. ume expansion; reference 2) stimulate thiazide-sensitive Na-Cl Ellison). cotransport. In the present experiments, I 1HSD2 expression levels ap- pear to be highest along the connecting tubule, where the References enzyme is expressed by cells that also express the Na/Ca I . Rossier BC, Palmer LG: Mechanisms of aldosterone action on sodium and transport. In: The Kidney: Physiology and exchanger. The concordance between sites of Na/Ca exchanger Pathophvsiologv, 2nd Ed., edited by Seldin DW, Giebisch 0, and I I HSD2 expression raises the possibility that mineralo- New York, Raven, 1992, pp 1373-1409 corticoid hormones affect transport in the distal tubule. 2. Vel#{225}zquez H, Bartiss A, Bernstein PL, Ellison DH: Adrenal Addison’s disease may present with hypercalcemia (48), and steroids stimulate thiazide-sensitive NaCl transport by the rat states of mineralocorticoid excess such as Conn’s syndrome renal distal tubule. Am J Physiol 39: F2l l-F2l9, 1996 may be associated with calcium wasting (49). The aldosterone 3. Chen Z, Vaughn DA, Blakeley P, Fanestil DD: Adrenocortical antagonist reduces renal calcium excretion (50). steroids increase renal thiazide receptor density and Although some studies suggested that mineralocorticoid hor- response. J Am Soc Nephrol 5: 1361-1368, 1994 mones do not have direct effects on calcium transport (5 1 ,52), 4. Plotkin MD, Kaplan MR. Verlander JW, Lee W-S, Brown D, the current studies raise the possibility that mineralocorticoid Poch E, Gullans SR. Hebert SC: Localization of the thiazide hormones may influence distal calcium transport. sensitive Na-Cl cotransporter, rTSC1 , in the rat kidney. Kidney list 50: 174-183, 1996 The present results and the results of others are summarized 5. ObermOller N, Bernstein PL, Vel#{225}zquez H, Reilly R, Moser D, in Table 1 . They indicate that high-level I 1HSD2 expression Ellison DH, Bachmann 5: Expression of the thiazide-sensitive begins along the DCI of rat, at the junction between the DCT Na-Cl cotransporter in rat and human kidney. Am J Phvsiol 269: I and DCI 2. 1 1-Hydroxysteroid dehydrogenase type 2 cx- F900-F9l0, 1995 pression is highest along the connecting tubule, remains high 6. Bachmann S. Vel#{225}zquez H, ObermUller N, Reilly RF, Moser D, along the cortical collecting duct, and declines as collecting Ellison DH: Expression of the thiazide-sensitive Na-Cl cotrans- tubules course into the medulla. Mineralocorticoid receptors porter by rabbit distal convoluted tubule cells. J Clin Invest 96: are expressed throughout the distal nephron, from the thick 2510-2514, 1995 J Am Soc Nephrol 9: 1347-1358, 1998 Cotransporter Expression by Distal Tubules I 357

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