Sgk1 Gene Expression in Kidney and Its Regulation by Aldosterone: Spatio-Temporal Heterogeneity and Quantitative Analysis
Jianghui Hou,
Helen J. L. Speirs,
Jonathan R. Seckl and
Roger W. Brown
Molecular Medicine Centre, Western General Hospital, Edinburgh, United Kingdom.
Correspondence to: Dr. Roger W. Brown, Molecular Medicine Centre, Western General Hospital, Edinburgh EH 2XU, United Kingdom. Phone: 44-131-651-1024/1037; Fax: 44-131-651-1085; E-mail: rbrown{at}srv0.med.ed.ac.uk
ABSTRACT. The serine-threonine kinase sgk1 was recently identifiedas a gene rapidly induced by mineralocorticoids, resulting inincreased sodium transport in vitro. To carefully localize andquantify the renal sgk1 expression response to aldosterone,in situ hybridization was performed on kidneys of mice havingaldosterone excess over a range of doses and durations. In controland adrenalectomized animals, the glomeruli and inner medullarycollecting ducts were the major sites of sgk1 expression, whichwas maintained independent of aldosterone. Sgk1 upregulationinduced by aldosterone excess exhibited spatio-temporal heterogeneity.Both acute (3-h) and chronic (6-d) aldosterone excess stimulatedsgk1 expression in the distal nephron, i.e., from the distalconvoluted tubules through to the outer medullary collectingducts. Treatments for 6 d with low sodium diet (0.03% [I]) andaldosterone infusions (50 µg/kg per d [II], 150 µg/kgper d [III], and 750 µg/kg per d [IV]) generated elevationof circulating aldosterone. Across these treatments (I throughIV), the circulating level correlated with the progressive inductionof sgk1 expression, with highly stimulated tubules first appearingin cortex (I) and continuing downward (II) until there was astrong stimulation throughout outer medulla (III and IV). Interestingly,chronic but not acute aldosterone excess caused a slight increaseof sgk1 expression in glomerulus (30 to 50%; P < 0.01) anda dramatic downregulation in the initial portion of inner medulla,which could result from diminished interstitial osmolarity.Relative quantification (versus control) of sgk1 upregulationin individual tubules revealed: (1) a 1.8-fold increase of sgk1mRNA at 3 h (150 µg/kg injection) and (2) a dose-dependenceof chronic upregulation reaching a ceiling of eightfold elevation.
Regulation of sodium reabsorption in kidney is crucial for aintenanceof whole body fluid homeostasis. The Na+ transport induced byaldosterone, either in vivo or in vitro, is sensitive to theK+-sparing diuretic amiloride, which is a potent blocker ofthe epithelial Na+ channel (ENaC) (1–3). The major siteof aldosterone action is in the cortical collecting duct, includingits initial portion. Receptor sites and metabolic effects ofmineralocorticoids have been documented through the second halfof distal convoluted tubules (DCT) and in the connecting tubulesand the medullary collecting ducts (4–5). Two phases aredistinguished in its regulation of sodium transport: an earlyphase starting after a lag period of 20 to 60 min, during whichthe preexisting channels and Na/K-ATPase pumps are activated,and a late phase (>2.5 h), which involves increasing contributionfrom newly synthesized channel and pump proteins (6).
In the A6 Xenopus cell line, which is a good model of mammaliandistal nephron, efforts have been made to elucidate the pathwayof aldosterone action, leading to the finding of an early-responsegene, sgk1 (serum/glucocorticoid kinase), the transcriptionof which is rapidly (<3 h) stimulated by aldosterone excess(7). Intriguingly, sgk1 was shown to significantly elevate ENaC-mediatedsodium transport via increasing the surface expression of ENaCsubunits in Xenopus oocytes (8), which is in keeping with evidencesuggesting sgk1 may facilitate a similar increased apical translocationof ENaC in distal nephron in vivo (9–10). Other stimuli,such as insulin, glucocorticoids, and cell volume change, weredocumented to regulate sgk1 (11–14). These in vitro findingsidentify sgk1 as a key gene in hormonal regulation of sodiumtransport in cells of distal nephron. It thus becomes of greatinterest to determine the distribution of sgk1 in intact kidneyand study its hormonal regulation in vivo.
Previous animal studies have been focusing on the short-term(0.5- to 4-h) effect of aldosterone on sgk1 expression (7,15–16).First, we aim to elucidate whether regulation of sgk1 expressionis consistent with playing a role in transduction of aldosterone-drivensodium reabsorption in distal nephron in both short-term (acute)and chronic hyperaldosteronism. Second, we look for evidenceof other potential regulators of and roles for sgk1 in kidney.In particular, sgk1 expression may exhibit an aldosterone dosedependence in such a way that a plateau or a peak occurs ata dose above which there is no further induction or even a falloff in the degree of sgk1 induction. This would be of particularinterest, as it may identify a response moderating aldosterone-sgk1-drivensodium retention and imply that such a mechanism contributesto escape from aldosterone-induced Na retention. Finally, thesedetailed studies on a wide range of aldosterone-related treatmentswill allow a clearer view of sgk1 expression and regulationalong the nephron and will help to guide future studies. Toaddress these issues, we performed in situ hybridization usingradiolabeled riboprobes, autoradiographic films, and silvergrain counting, which are well-established means of relativequantitation of expression level (17). This approach allowscomparison of sgk1 mRNA expression levels in renal regions andnephron segments.
Materials
Steroids, propylene glycol, DMSO, and molecular biology-gradechemicals were purchased from Sigma Chemical Company (Poole,Dorset, UK), 1-kb ladder DNA size markers and synthesized oligonucleotideswere purchased from Life Technologies/Life Technologies (Paisley,Renfrewshire, UK), ALZET 1007D osmotic mini-pumps and mice werepurchased from Charles River UK (Margate, Kent, UK), and 35S[UTP]was purchased from Amersham International (Little Chalfont,Buck, UK).
Animal Treatments
The animal work was approved by the local ethical committeefor the care of animals and was in accordance with the NIH Guidelinesfor the Care and Use of Laboratory Animals. Eight-week-old maleC57BL/6J mice (23.5 to 25 g) were segregated into ten groups(each n = 6) and caged in pairs within their own group (Table 1).All mice had free access to normal diet (0.3% sodium) andwater (with saline additionally available to adrenalectomizedmice). The bilateral adrenalectomy operations were performedunder gaseous anesthesia. Animals were killed by terminal anesthesia.The ALZET 1007D osmotic mini-pumps (preloaded with treatmentsor vehicle) were implanted subcutaneously to mice, generatinggroups having aldosterone infusions with various doses (50 µg/kgper d, 150 µg/kg per d, and 750 µg/kg per d) for6 or 21 d. To investigate the acute effects of aldosterone,150 µg/kg injections were given to mice 3 h before sacrifice.Additionally, one group of mice was kept on 0.03% low-sodiumdiet for 6 d. Table 1 gives details of these groups.
Plasma Aldosterone Assay
Blood samples were removed by cardiac puncture rapidly afterinitiation of terminal anesthesia and centrifuged at 4°Cfor 10 min. Plasma was separated and stored at -20°C. Aldosteronewas assayed using a 125I-RIA kit (DPC, Los Angeles, CA).
In Situ Hybridization
On the basis of mouse sgk1 sequence (accession number: AF139638),primers were designed to amplify a fragment encompassing a region(1091 to 1336 bp) that had no significant homology to othersequences. Flanking T3 and T7 phage polymerase consensus siteswere introduced by nested PCR as described previously (18).Single-stranded [35S]UTP-labeled RNA probes were generated usingthe required RNA phage polymerases. Six mice were examined ineach group. For each mouse, one sagittal cryostat section (10µm) was cut from intact kidney, which included cortexand outer and inner medulla. Sections were thaw-mounted onto3-amino propyltriethoxysilane-coated slides and stored at -80°C.4% paraformaldehyde was used for fixation, followed by acetylationand prehybridization at 50°C for 2 h. Hybridization with4 x 106 c.p.m [35S]UTP-labeled RNA probe per slide was performedat 50°C for 14 to 16 h and then followed by RNaseA treatmentand washes with a maximum stringency of 0.1xSSC at 60°C.This follows well-established methodology that reflects findingsthat show that these conditions create the high subsaturation/saturationriboprobe concentrations that provide specific radiolabeledprobe hybridization that reflects the amount of target RNA present.When quantitatively detected (film and dipped emulsion autoradiography)this then allows relative quantitation for sgk1 expression betweenslides processed together. After washes, slides were dehydratedand placed against -Max Hyperfilms. Three to five days of exposuregave a satisfactory range of autoradiographs in the linear rangeof the films as judged by coincident exposure of the film toradioactive microscale standards for this purpose (calibratedfor moderate -emitters [14C, 35S] in thin tissue slices: #RPA504,RPA511; Amersham International, Little Chalfont, UK). All slideswere then dipped in photographic emulsion and exposed in a light-tightbox for 2 to 3 wk before being developed. This optimal timewas determined by the optimal exposure for the film autoradiographs.In preliminary work, we emulsion-dipped slides with attachedmicroscales (such as above) and found a wide linear range (correlation,r = 0.98), relating grain-count density versus radioactivity.For all the dipped slides in these sgk1 studies, grain-countdensities did not exceed this linear range. Selected sectionswere then counterstained with the periodic acid-Schiff (PAS)method.
Quantitative Image Analyses
Autoradiographic films were scanned on a high-resolution flatbedscanner. Developed emulsion-dipped slides were analyzed on aZeiss Axioskop Microscope (Carl Zeiss, Thornwood, NY) with theKS300 (v3) silver-grain counting software. Steps to minimizebackground tissue hybridization included the employment of apostfixing acetylation procedure to block the positively chargedammonium groups on tissue and a posthybridization RNase A treatmentto cleave unhybridized single-stranded RNA molecules. Thus,the tissue background for antisense probe was considered asthe nonspecific binding to tissue RNA by probes with equal GCpercentage, and so equal to the hybridization level on sensecontrol sections. When silver grains were counted in high magnificationfields, the tissue background was normalized for each slideas follows. The background grain density of tissue and glasson the sense control slide were measured separately. The ratiobetween such paired background measurements was calculated forthe slide. The tissue background for an antisense slide wasthus estimated by multiplying its glass background by this ratio.Quantification was done by counting the number of silver grainsover areas under high-power magnification (x400). Grain-countinginvolved determining the grain density over glomeruli and tubulesthat highly expressed sgk1. Identification of glomeruli wasstraightforward for all groups. Identification of tubules inboth cortex and outer medulla involved comparing highly expressingtubules in animals with aldosterone excess and those in controls,where tubular expression merged with "background" levels. Althoughthere was some variation across non-glomerular cortex (mean± SE grain density, 0.0869 ± 0.0106 counts/µm2),this did not clearly correspond to tubule outlines and was presentover a wider cortical area in control and treatment groups thanthe highly expressing tubules readily identifiable in treatmentgroups. The best valid solution to estimate control tubulargrain counts was to measure this grain density over a largertubular area, (full-field [x400] view of medulla or non-glomerularcortex) rather than individual tubules. This control expressionlevel was used for comparison with animals with aldosteroneexcess. We followed a protocol for random selection of regions,glomeruli, and tubules, which involved aligning the field ofview over renal cortex at such low power that the viewer wasunable to see local renal structure or grain density. The viewwas then zoomed to x100 to x200, and glomeruli or tubules inthe central field of view were those designated to be subjectedto grain counting at x400. The process was repeated, countingten glomeruli or tubules (with grain-density elevated allowingthem to be demarcated against background) out of eight sampledfields in each region (for cortex and medullary regions separately).Sampling cortical or medullary regions was carried out in thesame fashion for each kidney section. When it came to controlsections, five full-field (x400) views were counted (minus glomeruliin cortex, five rather than eight because of their larger area).Rarely, part of the section with poor morphology was excludedfrom subsequent statistical analyses. For each mouse, the meandensity of each such renal component assessed (glomeruli andcortical and medullary tubules or areas) was then calculated.
Statistical Analyses
The significance of differences between groups was tested byANOVA. When the all-effects F value was significant (P <0.05), post hoc analysis of differences between individual groupswas made with the Neuman-Keuls test. Values were expressed asmean ± SE unless otherwise stated.
Renal Expression Profile of sgk1
Examination of control and adrenalectomized animals revealedthat sgk1 mRNA had a discrete distribution in kidney, with apunctate appearance in cortex, little expression in outer medulla,and high abundance in inner medulla (Figure 1A). This expressionprofile was not dependent on circulating corticosteroids, asit was maintained in adrenalectomized animals (aldosterone <400± <100 pmol/L; corticosterone <55 nmol/L). Thecortical expression of sgk1 was primarily confined to glomeruli(Figure 1B). Its expression in glomeruli was uniform and centrallydistributed (Figure 1E), ruling out the possibility that theglomerular visceral or parietal epithelium was the expressionsite. This is consistent with the data from human kidney (19),suggesting sgk1 expression in the glomerular mesangium. In innermedulla, nearly all cells strongly expressed sgk1 as the papillawas approached (Figure 1, C and D). This characteristic indicatesthat the inner medullary collecting ducts (IMCD) are the expressionlocation, because the number of thin limbs of Henle’sloop diminishes toward papilla.
Figure 1. Sgk1 expression profile in kidney. Autoradiograph (A: expression = black) and photomicrographs (B through D: dark-field, expression = white; E: bright-field, expression = visible silver-grains) showing sgk1 expression in detail in the kidney of a normal (control) mouse. The boxes in the autoradiograph in panel A show the sites of capture of the photomicrographs on the corresponding kidney section slide (B: x100, cortex; C and D: x50, medulla). A single glomerulus was visualized in panel E at x400 magnification. Note the abundant expression in glomeruli and inner medulla. Glom, glomerulus; Cx, cortex; oM, outer medulla; iM, inner medulla.
Chronic Effects of Aldosterone and Dosage Response
A range of 6-d treatments with sodium depletion (I: 0.03% sodiumdiet) and aldosterone infusions (II through IV: 50 µg/kgper d, 150 µg/kg per d, and 750 µg/kg per d) resultedin sustained elevation of circulating aldosterone levels: I,1234 ± 124 pmol/L; II, 4660 ± 120 pmol/L; III,15485 ± 471 pmol/L; and IV, 53569 ± 3198 pmol/L.In view of the fact that severe sodium deficiency has been reportedto increase aldosterone secretion 20- to 25-fold in sheep orhuman (20–21), the two high-dose infusions (III and IV)mimicked these physiologically extreme conditions of aldosteroneexcess (25-fold and 75-fold elevation of plasma level versus699 ± 56 pmol/L of controls). Correlated with the elevationof circulating aldosterone across these animal groups, therewas a change in the pattern of sgk1 expression, with highlystimulated tubules first appearing in cortex (I, Figure 2, A and a)and continuing downward (II, Figure 2, B, b, and c) untilthere was a strong stimulation throughout outer medulla (IIIand IV, Figure 2, C and d). Silver-grain counting of individualtubules showed a dose-dependent upregulation of sgk1 in bothcortex and outer medulla (Figure 3). The mild excess of aldosteronein sodium-depleted animals (I) induced a 4.7-fold increase ofsgk1 expression, which was confined to cortical tubules. Suchcortical induction reached the ceiling (eightfold) in animalsinfused with aldosterone (II and IV). In outer medulla, sgk1stimulation (6.4-fold) was first observed in animals with moderatealdosterone excess (II). The ceiling of eightfold increase wasreached at groups with severe aldosterone excess (III and IV).Moreover, in animals treated with 150 µg/kg aldosteronefor 21 d, sgk1 stimulation persisted and its pattern and extentremained similar to that at 6 d (Figure 3). Finally, water intakeof animals was substantially increased by the high-dose aldosteroneinfusions (twofold in the 150 µg/kg per d groups and threefoldin the 750 µg/kg per d group versus control).
Figure 2. Chronic effects of aldosterone on sgk1 expression. Autoradiographs (A through C: expression = black) and photomicrographs (a through e: dark-field, expression = white) showing sgk1 expression in kidneys of animals treated with aldosterone excess (Magnifications: x100 in a and b; x50 in c through e). The sites of capture of the photomicrographs on the kidney sections are indicated on the corresponding autoradiographs. Note the highly stimulated tubules traversing from cortex to outer medulla. T, stimulated tubules.
Figure 3. Quantification of sgk1 expression in response to chronic aldosterone excess. Mice were treated for 6 d with sodium depletion, low-dose (50 µg/kg) and high-dose (150 to 750 µg/kg) aldosterone infusions or for 21 d with 150 µg/kg infusion. Sodium depletion induced a 4.7-fold increase of sgk1 mRNA level in cortical tubules (P < 0.01 versus control). The 50 µg/kg infusion stimulated sgk1 by 8.2-fold in cortex and 6.4-fold in outer medulla. Sgk1 stimulation by high-dose infusions for 6 or 21 d reached a ceiling of upregulation (eightfold elevation) in cortex and outer medulla. The mean grain density in cortical tubules of control animals was designated as one unit.
Acute Effects of Aldosterone
In mice injected with 150 µg/kg aldosterone 3 h beforesacrifice, highly stimulated tubules were visualized in cortexand outer medulla (Figure 4). The rapid sgk1 induction causedby injection was completely abolished in animals pretreatedwith spironolactone (MR antagonist) (Figure 4), suggesting mediationvia mineralocorticoid receptors (MR). Silver-grain countingto examine the acute effects of aldosterone revealed a 1.8-foldupregulation of sgk1 in cortical tubules and 1.4-fold in outermedullary tubules, lower than that elicited by the chronic treatments,e.g., 6 d and 21 d (Figure 5). Nevertheless we cannot rule outthe possibility that a peak of sgk1 stimulation may have occurredbefore 3 h, considering the short half-life of circulating aldosterone(20 min) (22) and sgk1 mRNA (30 min) (23). Furthermore, otherstudies showed that sgk1 stimulation peaked in rat kidney within1 to 2 h after aldosterone injection (15–16).
Figure 4. Acute effects of aldosterone on sgk1 expression. Autoradiographs (expression = black) showing comparison of sgk1 expression among control (A), animals injected with 150 µg/kg aldosterone 3 h before sacrifice (B), and those pretreated with spironolactone (MR antagonist) (C).
Figure 5. Quantification of sgk1 expression in response to acute aldosterone excess. Aldosterone-induced upregulation of sgk1 mRNA by 1.8-fold in cortex and 1.4-fold in outer medulla (P < 0.01 versus control). The mean grain density in cortical tubules of control animals was designated as one unit.
Aldosterone Induction of sgk1 Expression Is in Distal Nephron
Examination of emulsion-dipped slides revealed both acute andchronic aldosterone treatments induced sgk1 expression in aminority of cortical tubules. The periodic acid-Schiff counterstainingmethod stains the brush border of proximal tubules with pinkcolor. Sgk1 stimulation was visualized in tubules lacking thisborder (Figure 6, A, B, D, and E), indicative of more distaltubules, which in cortex were distributed widely across corticallabyrinths as well as in cortical medullary rays; in fact, fromDCT to cortical collecting ducts (CCD). Moreover, tubules atthe vascular pole of glomerulus exhibited sgk1 induction (Figure 6, C and F),suggesting possible involvement of the early portionof DCT. However to characterize the DCT distribution definitivelywould require additional studies. Sgk1 stimulation could beseen in long single tubules extending in continuity from cortexthrough outer medulla to enter inner medulla without a majorchange in diameter (Figure 2, c through e). This pattern wasfully consistent with the cortical and outer medullary collectingducts (OMCD) being sites of upregulation of sgk1 expression.
Figure 6. Histology of renal cortical sections from aldosterone-treated groups. The images (magnification, x100) of panels A through C were taken by phase-contrast microscopy, and D through F are their respective counterparts under dark-field view. The cortical labyrinth (CL) is illustrated on panel A. Panel B shows a region where a cortical medullary ray (CMR) protrudes into cortical labyrinth. Periodic acid-Schiff (PAS) stains the brush border of proximal tubules (PT). In dark-field view (D through F), the counterstaining caused the image to have a dark green (negative) or dark purple (positive) background, on top of which silver grains are visible. In cortex (both CL and CMR), the tubules in which upregulation of sgk1 by aldosterone is seen are PAS-stained negative, indicating they are tubules of the distal nephron. Glomerulus and surrounding structures are illustrated in panels C and F. The distal tubule approximating to the vascular pole of glomerulus exhibited sgk1 induction, suggesting the possible involvement of the early portion of distal convoluted tubules. Glom, glomerulus; VP, vascular pole; UP, urine pole; +, high expression.
Distinct Regulation by Aldosterone in Glomeruli and IMCD
It was somewhat unanticipated that sgk1 mRNA abundance was raised(30 to 50%) by chronic (50 µg/kg per d) but not acutealdosterone excess in glomeruli (Figure 7), a site not regardedas a classical MR-responsive target. A similar upregulationwas also detected in animals with higher doses of aldosteroneinfusion but not in those with sodium depletion. Interestingly,the abundant sgk1 expression in inner medulla was downregulatedin its initial portion by chronic and severe aldosterone excess(150 or 750 µg/kg per d for 6 or 21 d). The degree ofthis effect showed some individual variation within treatmentgroups and was more striking at 21 d. As illustrated in Figure 2, C and e,the outer-inner medulla junction was easy to identifyon the autoradiograph, where the OMCD with high sgk1 expressionwere adjoining the inner medulla with much repressed expression.
Figure 7. Quantification of sgk1 expression in glomerulus. Silver-grain counting revealed a slight increase (30 to 50%) of sgk1 expression induced by chronic (6 d) but not acute (3 h) aldosterone excess (* P < 0.01 versus control).
The expression of sgk1 mRNA and its regulation by aldosteronewere studied in detail. In kidney of control mice, we foundan abundant expression of sgk1 in glomeruli and IMCD, whichwas substantially maintained independent of corticosteroidsand thus persisted in adrenalectomized animals. In mice, bothacute and chronic aldosterone infusions induced sgk1 expressionin distal tubules through to OMCD. These regions have the potentialto mediate amiloride-sensitive sodium transport, as they arethe major sites of ENaC action. Although sgk1 was shown to increasethe surface expression of ENaC subunits in Xenopus oocytes (8),a recent report suggested that ENaC translocation to the apicalmembrane was limited to the proximal portion of the distal nephron,i.e., from DCT to connecting tubules, in response to rapid aldosteroneinfusion (2 to 4 h) in rats (9). Both our findings (mRNA) andreports of other groups (protein) showed that acute sgk1 accumulationwas localized to the entire aldosterone-sensitive distal nephron(ASDN: i.e., from DCT to OMCD), an axially heterogeneous mechanismcan be proposed to control the activation of sgk1 (e.g., byphosphorylation) (9), and thereby restrict ENaC translocationto the more upstream portion of the distal nephron. Beyond previousfindings, our work shows an axial heterogeneity of prolongedupregulation of sgk1 mRNA, which correlates with the plasmalevel of aldosterone across groups on low-sodium diet to low-doseand high-dose aldosterone infusions. It appeared that mild aldosteroneexcess (0.03% sodium diet) induced sgk1 in the cortical portionof ASDN, whereas moderate (50 µg/kg infusion) and moresevere (150 µg/kg and 750 µg/kg infusions) excessaffected the entire ASDN. Two recent studies in rat (10) andmouse (24), have reported that treatments with chronic aldosteroneexcess (10) or dietary sodium restriction (24) are associatedwith ENaC translocation in distal nephron. Dietary sodium restrictionin mice caused ENaC to shift from cytoplasm to the apical membranein cells confined to the cortical segments of ASDN (24). Thiscould be interpreted as potentially due to the accumulationof sgk1, which linked aldosterone effects to ENaC insertion.
This study is the first to assess sgk1 regulation using in situhybridization in mouse as well as the first to carefully quantifythe degree of its induction in different parts of the nephron(distal tubules, glomeruli, and inner medulla) using silver-graincounting. Previous work has examined aldosterone regulationof sgk1 mRNA levels in renal cell lines (7,25), the whole kidney(15–16), or dissected renal components (9,26) by northernblotting or RT-PCR and has demonstrated the accumulation ofsgk1 protein in response to hormone excess by immunohistochemistry(7,9). As the current work examines the effects of a wider rangeof treatments (Table 1), this allows a clearer view of the wayaldosterone affects sgk1 expression across the nephron. In thepresent study, up to an eightfold increase of sgk1 mRNA wasfound in individual renal tubules in response to chronic aldosteroneexcess and 1.8-fold to acute excess. The upregulation of sgk1mRNA exhibited dose dependence over a range of circulating aldosterone(2- to 75-fold elevation). The reaching of its ceiling (eightfoldelevation) in animals with severe aldosterone excess suggestedthe saturation of available MR sites and of sgk1-mediated sodiumretention via ENaC.
The mediator that upregulated sgk1 in glomeruli remains unclear.It is possible that aldosterone at high doses will bind to glucocorticoidreceptor to some degree and subsequently induce sgk1 expression(27–28). However, circulating physiologic glucocorticoidlevels (18) supply a better glucocorticoid receptor ligand ateven higher levels than the raised plasma aldosterone in ouraldosterone excess groups, especially the 50 µg/kg perd group, which exhibits this glomerular sgk1 upregulation. Moreoveradrenalectomy does not abolish glomerular sgk1 expression, indicatingthat other pathways are involved. As elevated mesangial sgk1in diabetic nephrons has been thought to follow altered GFR(19), it may be that glomerular hemodynamic changes during chronicaldosterone excess initiate the mesangial sgk1 upregulation.
The axial osmolality gradient in the renal medulla is made upof gradients of several individual solutes, including NaCl andurea. In antidiuresis, progressive increases in tissue NaCland urea concentration and osmolality are observed along thecorticomedullary axis from the cortex to the papilla. In contrast,the gradients are markedly attenuated during water diuresis(29). We propose an osmotic theory to explain the high abundanceof sgk1 expression in IMCD, although we cannot rule out thepossibility of sgk1 expression in interstitial cells. Evidencefrom other research supports this proposal and it seems thatthe extracellular hypertonicity or cell shrinkage can stimulatesgk1 expression in vitro (14). Thus, a certain osmotic thresholdsufficient to induce sgk1 may be reached at the outer-innermedullary junction. The unexpected downregulation of sgk1 ininner medulla induced by severe aldosterone excess indicatesthe engagement of regulatory mechanisms other than MR mediation.It is possible that the osmotic gradient was partially washedout by the increased water intake (twofold in the 150 µg/kgper d groups and threefold in the 750 µg/kg per d groupversus control), which was elicited by long-term and severealdosterone excess. Hence, in such a partial "wash-out," theosmotic gradient including the threshold at which sgk1 was stimulatedwould move further distally toward papilla; this may explainthe reduction of sgk1 expression in the initial part of innermedulla. Although further investigation is required, as thereis an individual variation in fluid intake with chronic andsevere aldosterone excess, it may account for the correspondingvariation in the degree to which inner medullary sgk1 expressionis downregulated under these circumstances. The action of sgk1in inner medulla is unclear, but it could be involved in controllingcell volume in the face of high interstitial osmolarity towardthe papilla. Additionally, it could be involved in regulatingsodium reabsorption in IMCD. In the latter case, a reductionin sgk1 expression with prolonged aldosterone infusion mightcontribute to facilitating natriuresis in "escape" from thesodium retention induced by aldosterone.
Acknowledgments
We thank the University of Edinburgh Medical School and theUK Overseas Research Scheme (jointly supported studentship toJH); and the Scottish Hospital Endowment Research Trust (grantRg77/00), British Heart Foundation (grant FG/2001075), and UrquhartCharitable Trust for their support. We thank June Noble, LynneE. Ramage, and Lawrence Brett for their expert technical assistance.We thank Dr. Chris Kenyon for his help and valuable advice onthe aldosterone and corticosterone assays.
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Received for publication August 3, 2001.
Accepted for publication December 22, 2001.
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Top
Abstract
Introduction
-Max Hyperfilms. Three to five days of exposure gave a satisfactory range of autoradiographs in the linear range of the films as judged by coincident exposure of the film to radioactive microscale standards for this purpose (calibrated for moderate 
, 
subunit proteins in rat kidney. J Clin Invest 104: R19, 1999[Medline]
