Increased renal -ENaC and NCC abundance and elevated blood pressure are independ - 《American Journal of Physiology》美国生理学杂志干细胞之家



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发表于 2009-4-22 08:40 |只看该作者 |倒序浏览 |打印

作者：Swasti Tiwari, Randall K. Packer, Xinqun Hu, Yoshihisa Sugimura, Joseph G. Verbalis,, and Carolyn A. Ecelbarger,作者单位：1 Division of Endocrinology and Metabolism, Department of Medicine, and 2 Center for Sex Differences, Georgetown University, Washington; and 3 Department of Biological Sciences, George Washington University, Washington, District of Columbia

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      【摘要】
   Previously, we demonstrated that rats undergoing vasopressin escape had increased mean arterial blood pressure (MAP), plasma and urine aldosterone, and increased renal protein abundance of the -subunit of the epithelial sodium channel (ENaC), the thiazide-sensitive Na-Cl cotransporter (NCC), and the 70-kDa band of -ENaC (Song J, Hu X, Khan O, Tian Y, Verbalis JG, and Ecelbarger CA. Am J Physiol Renal Physiol 287: F1076-F1083, 2004; Ecelbarger CA, Knepper MA, and Verbalis JG. J Am Soc Nephrol 12: 207-217, 2001). Here, we determine whether changes in these renal proteins and MAP require elevated aldosterone levels. We performed adrenalectomies (ADX) or sham surgeries on male Sprague-Dawley rats. Corticosterone and aldosterone were replaced to clamp these hormone levels. MAP was monitored by radiotelemetry. Rats were infused with 1-deamino-[8- D -arginine]-vasopressin (dDAVP) via osmotic minipumps (5 ng/h). At day 3 of dDAVP infusion, seven rats in each group were offered a liquid diet [water load (WL)] or continued on a solid diet (SD). Plasma aldosterone and corticosterone and urine aldosterone were increased by WL in sham rats. ADX-WL rats escaped, as assessed by early natriuresis followed by diuresis; however, urine volume and natriuresis were somewhat blunted. WL did not reduce the abundance or activity of 11- -hydroxsteroid dehydrogenase type 2. Furthermore, the previously observed increase in renal aldosterone-sensitive proteins and escape-associated increased MAP persisted in clamped rats. The densitometry of immunoblots for NCC, - and -70 kDa ENaC, respectively, were (% sham-SD): sham-WL, 159, 278, 233; ADX-SD, 69, 212, 171; ADX-WL, 116, 302, 161. However, clamping corticosteroids blunted the rise at least for NCC and -ENaC (70 kDa). Overall, the increase in aldosterone observed in vasopressin escape is not necessary for the increased expression of NCC, - or -ENaC or increased MAP associated with "escape."
      【关键词】 SIADH adrenalectomy mean arterial pressure natriuresis hyponatremia5 e: i+ _" F4 r7 S6 r( U' }
   VASOPRESSIN IS A MAJOR HORMONAL regulator of body water homeostasis. Vasopressin levels are inappropriately high in the syndrome of inappropriate antidiuretic hormone (ADH) secretion (SIADH). This syndrome is characterized by water retention and hyponatremia, potentially serious clinical disorders observed in hospitalized patients ( 30 ). The water-retentive phase is generally relieved by a physiological process known as "vasopressin escape," in which both humans and animal models undergo early natriuresis followed closely by a diuresis of increasingly more dilute urine. The mechanism and trigger of both the diuresis and natriuresis are not fully understood, although increased renal arterial pressure appears essential for the process ( 16 ).

It is now known that the "escape" from the antidiuretic action of ADH or vasopressin is facilitated by downregulation of aquaporin-2 protein (AQP-2), a water channel, in the kidney collecting duct (CD) ( 12 ). Downregulation of AQP-2 is associated with reduction in CD osmotic water permeability and hence is the most likely mechanism by which diuresis occurs ( 7 ).% k  g" F# D+ A" h; e( `

Sodium balance is maintained by the regulation of sodium reabsorption in the distal convoluted tubule (DCT) and CD. The thiazide-sensitive (Na-Cl) cotransporter (NCC) of the DCT and the epithelial sodium channel (ENaC) of the late DCT through the CD are important apical sodium transport pathways. We previously showed that these aldosterone-sensitive renal proteins are markedly upregulated in escaping rats ( 10, 29 ). This increased sodium-reabsorptive capacity of the distal tubule might be protective in ameliorating the hyponatremia during vasopressin-induced water retention.  i# N# }# Z7 O1 Z

The regulation of sodium-reabsorptive pathways during vasopressin escape at the molecular level is not fully understood. Aldosterone, a major hormonal regulator of sodium reabsorption in the distal tubule, increases the abundance of NCC ( 21 ) as well as the -subunit of the ENaC ( 24 ). However, the manner in which the renin-angiotensin-aldosterone axis responds during SIADH or in animal models of vasopressin escape is not entirely clear. Overall, an increase in aldosterone levels or a relative increase in mineralocorticoid receptor signaling would be predicted to increase sodium transport in these renal segments. Recently, we reported increased urine and plasma aldosterone in escaping rats along with the increase in these renal proteins and a rise in blood pressure ( 29 ). However, it was not clear whether the elevated aldosterone was the cause of these increases or just an associated phenomenon.6 \( M1 A/ C. w, p6 I3 J; b
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This study was carried out to determine whether the increase in aldosterone was essential for the renal protein changes and "escape-associated" increased blood pressure. To answer this question, we performed adrenalectomies (ADX) on male Sprague-Dawley rats and replaced the level of aldosterone and corticosterone at a clamped but "physiological" level. We decided to replace these two primary corticosteroids at a physiological level, rather than study the absolute absence of these hormones, as we were aware of the critical need for at least corticosterone in maintaining normal glomerular filtration rate ( 1, 15 ), which could clearly affect vasopressin escape. We then examined the abundance of aldosterone-sensitive renal proteins and blood pressure during vasopressin escape.

MATERIALS AND METHODS& G# y7 r# c( [1 m# k" Q
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Animals and study design. For this study, male Sprague-Dawley rats, 275 g, were obtained from Taconic Farms (Germantown, MD). Rats were divided into the following four treatment groups ( Fig. 1 ). 1 ) Sham ADX desmopressin (1-deamino-[8- D -arginine]-vasopressin; dDAVP) infused solid diet (SD), n = 5; 2 ) sham ADX dDAVP infused feeding liquid diet (WL), n = 7; 3 ) ADX replacement (35 mg pellet of corticosterone, 1.5 µg·100 g body wt -1 ·day -1 aldosterone infusion) dDAVP SD, n = 6; and 4 ) ADX replacement dDAVP WL, n = 7. All rats were anesthetized with isoflurane (IsoFlo; Abbot Laboratories, North Chicago, IL) for all surgeries. Before adrenalectomies or sham surgery, radiotelemetry transmitters (Data Sciences International, St. Paul, MN) were inserted into a subset of 12 rats as previously described ( 29 ). Three rats with radiotelemetry transmitters were assigned to each of the abovementioned groups. After 7 days, adrenalectomies (ADX, n = 13) or sham ( n = 12) operations were performed on all rats, and corticosteroids were replaced in ADX rats by implantation of a subcutaneous 21-day release, 35 mg pellet of corticosterone, and an osmotic minipump (model 2002; Alzet, Cupertino, CA) to infuse aldosterone at the rate of 1.5 µg·100 g body wt -1 ·day -1. On the same day dDAVP-infusing osmotic minipumps were inserted into a second subcutaneous pocket to infuse at a rate of 5 ng/h into all rats. DDAVP (Aventis Pharmaceuticals, Bridgewater, NJ) is a selective vasopressin V 2 receptor agonist. At this time, all rats were fed a dry, pelleted AIN-76 formulation diet (BioServe, Frenchtown, NJ). After 3 days, rats were further subdivided into two treatment groups: SD and WL. Rats in sham-WL ( n = 7) and ADX-WL ( n = 7) groups were switched to a liquid AIN-76 diet containing a high amount of water (80% by weight). The remaining rats were continued on the dry, pelleted diet and water ad libitum [sham-SD ( n = 5) and ADX-SD ( n = 6) group] for 7 additional days. Rats were housed continuously in Nalgene metabolic cages (Harvard Apparatus, Holliston, MA) to facilitate urine collection. In addition, the volume of liquid diet consumed by the WL-treated groups was measured and recorded each day. Each day, any remaining liquid diet was discarded, liquid diet drinking dispensers were replaced or cleaned, and fresh liquid diet was added to avoid bacterial spoilage. With the use of the radiotelemetry system, blood pressure was measured for 10 s at 10-min intervals for the entire study. All animals were maintained at all times under conditions and protocols approved by the Georgetown University Animal Care and Use Committee, an American Association for Accreditation of Laboratory Animal Care-approved facility. Finally, all rats were killed by decapitation, and both heparinized and K -EDTA blood was collected for analyses. The right kidney was removed rapidly and frozen at -80°C for immunoblotting analyses.
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Fig. 1. Schematic diagram of study design.

Urine and plasma analyses. Urine was analyzed for Na  , K (ion-selective electrode system, EL-ISE electrolyte system; Beckman Instruments, Brea, CA), osmolality (freezing-point depression, The Advanced Osmometer model 3900; Advanced Instruments, Norwood, MA), and aldosterone (Coat-a-Count RIA kit; Diagnostic Products, Los Angeles, CA). Whole blood was centrifuged at 3,000 rpm (Sorvall RT 6000 D; Sorvall, Newtown, CT) at 4°C for 20 min to separate plasma. Plasma was analyzed for Na  , K  , osmolality, aldosterone, and corticosterone (RIA; Diagnostic Products).* s' l9 s( `5 w/ J. x# P. x) |' z

Immunoblotting. Whole kidney homogenates were prepared as previously described ( 9, 10 ). Initially, Coomassie-stained "loading gels" were done to assess the quality of the protein by sharpness of the bands and to confirm equality of loading, as previously described ( 8, 10 ). For immunoblotting, 20-30 µg protein from each sample were loaded into individual lanes of minigels of 7 or 10% polyacrylamide (precast; Bio-Rad, Hercules, CA). Blots were probed with our own polyclonal antibodies against -, and -subunits of ENaC, with polyclonal antibodies against NCC, -ENaC, and AQP-2, a kind gift from Dr. M. A. Knepper, NIH, and NKCC2 polyclonal antibody, a kind gift from Dr. J. Klein, Emory, University. AQP-3 polyclonal and 11- -hydroxysteroid dehydrogenase (type 2; 11 HSD2) polyclonal antibodies were obtained from Calbiochem (San Diego, CA) and Chemicon (Temecula, CA), respectively. All antibodies have been previously characterized ( 13, 14, 18 - 21, 24 ).2 a/ Y& f# {% V5 y6 m

11 -hydroxysteroid dehydrogenase type 2 activity. Microsomes were isolated from whole kidney homogenates. Kidneys were homogenized in 0.1 M sodium phosphate buffer (pH 7.4, 25 ml buffer per gram of tissue), containing 0.25 M sucrose. Microsomes were extracted by differential centrifugation at 10,000 g for 30 min; the supernatant was then centrifuged at 105,000 g for 60 min. The pellets obtained were resuspended in phosphate buffer and stored in liquid nitrogen.5 `8 q( M, ]* `+ H6 W0 J

To measure enzyme 11 -hydroxysteroid dehydrogenase type 2 (11 -HSD2) activity, 5 µg of protein of the thawed microsome preparations were added to 0.5-ml incubation solution (50 mM Tris·HCl, 1 mM MgCl 2, 10 mM DTT, and 0.5 mM NAD) plus 200,000 dpm ( 1, 2, 6, 7 )- 3 H corticosterone and samples were incubated at 37°C for 15 min. Controls lacking microsomes were also incubated as above. Incubations were terminated by addition of 3 ml dichloromethane. Samples were dried under air and then reconstituted with 50 µl dichloromethane, spotted onto thin-layer chromatography (TLC) plates along with unlabeled corticosterone and 11-dehydrocorticosterone, and separated in acetone-dichloromethane (18:82, vol/vol). Bands on the TLC plate containing corticosterone and 11-dehydrocorticosterone were located using a 250 nM UV light and were scraped from the plates and eluted into 1 ml isopropanol. Samples were counted by scintillation spectrophotometry using a Beckman model LS 6500 scintillation counter. Measurements were made in duplicate. Relative conversion was determined by the cpm for the 11 dehydrocorticosterone relative to starting material, i.e., sum of cpm for corticosterone and 11 dehydrocorticosterone.; u; D7 E7 D, w. Z5 x% E

Statistics. To determine differences between specific mean pairs, data were analyzed by one-way ANOVA followed by Tukey's multiple comparisons test or Kruskal-Wallis ANOVA on ranks followed by Dunn's multiple comparisons test (when data were not normally distributed or variance was different between groups). Multiple comparisons tests were only applied when a significant difference was determined in the ANOVA analysis, P 9 x" W% \% p+ d
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Liquid diet intake. To determine whether the adrenalectomy with clamped steroid levels affected the intake of the liquid diet, daily volume consumed was measured and recorded. In Fig. 2, we show calculated calories consumed by the WL rats in both treatment. As we observed before ( 25 ), water loading initially results in a marked decrease in diet consumed, with a rebound as rats escape. No difference, however, was observed between the two WL groups.1 X, l+ g1 ?7 Z7 f9 K3 w( n7 X  a0 m

Fig. 2. Kilocalorie intake from liquid diet. Volume of liquid diet consumed was measured daily in water load (WL) groups and gross calories consumed was calculated based on the dietary formulation provided by the vendor ( n = 7/group). Mean daily intakes are shown for 3 consecutive 3-day periods of the study., Sham-WL;, adrenalectomized (ADX)-WL. No significant difference between these 2 groups was observed.

Urinary aldosterone. Figure 3 shows urine aldosterone excretion in rats in this study. Urine aldosterone was low and not different among all treatments before the beginning of water loading ( day -1 ). It increased markedly in the sham water-loaded rats (sham-WL) to a level of 560% of that in the sham-SD rats after 5 days of water loading. However, there were no significant differences between ADX-WL and ADX-SD groups, indicating successful abolition of the rise in aldosterone in these WL rats. ADX rats of both treatment groups tended to have higher urine aldosterone excretion relative to sham-SD rats both before and after the water load, suggesting that we were replacing at slightly higher than the endogenous level; however, these differences were not significant.

Fig. 3. Aldosterone excretion at day -1 before water loading (filled bar), and at day 5, after water loading (gray bar). Before water loading, urine aldosterone was low and not different among the groups. Water loading significantly increased aldosterone excretion in sham-WL rats; however, it remained low in ADX-WL group. *Significant difference from sham-solid diet (SD) as determined by 1-way ANOVA.
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Verifying escape. Urine volume is plotted in Fig. 4 A. Similar to previous studies ( 7, 10, 12, 29 ), rats began to "escape" from the antidiuresis of dDAVP by day 2 or 3 after initiation of water loading. On day 1, the urine volume is significantly lower in ADX rats ( P = 0.012, by 2-way ANOVA, not indicated in figure). Urine volumes remain significantly higher in water-loaded groups from day 2 (P 0.05, by 2-way ANOVA). However, ADX-WL rats tended to have a blunted escape, with regard to urine volume, compared with sham-WL rats. However, there were no significant differences between any of the groups due to high variability.
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Fig. 4. Urine volume and osmolality during vasopressin escape. A : urine volume over the course of the experiment with WL commencing on day 0. Urine volume was increased significantly in the water load groups relative to the SD group by 3 days. B : urine osmolality over the course of the experiment. Osmolality was decreased significantly by water loading in both sham-WL and ADX-WL relative to SD groups (sham-SD and ADX-SD). Tukey's multiple comparisons test was applied only when 1-way ANOVA detected a significant ( P / S+ t& b* p- p' M9 f' t3 c
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Even before an increase in urine volume was observed, urine osmolality ( Fig. 4 B ) was reduced significantly in the rats undergoing escape, i.e., on day 1, relative to solid diet-treated rats ( P 0.015). Furthermore, this fall was more pronounced in the ADX-WL rats with ADX-WL rats being significantly different from the other three groups on day 1.1 y5 ~9 `- F7 @" D
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Natriuresis and urine K -to-Na ratio. Urine sodium is shown in Fig. 5 A. A marked natriuresis began at day 2 of water loading ( P 0.015, by 2-way ANOVA) as observed previously ( 10, 29 ) in both ADX-WL and sham-WL groups. Urine sodium was significantly reduced on day 1 in the ADX rats, relative to sham.( u6 u9 b+ G% ~# d
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Fig. 5. Urine Na excretion during vasopressin escape. A : water loading led to a marked natriuresis that peaked on day 2. B : urine K -to-Na ratio. The K -to-Na ratio was plotted for each day after water loading (WL), it is measured as an index of the renal aldosterone activity. This ratio was significantly suppressed from day 2 of water loading. After day 3 the ratio started increasing, indicating increasing Na retention relative to K during escape. Tukey's multiple comparisons test was applied only when 1-way ANOVA detected a significant ( P 2 x. G' ^& z4 i( Q( V. i
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Urine K -to-Na ratio is plotted ( Fig. 5 B ). This ratio should increase, independently of feed intake, when aldosterone activity is high in the kidney ( 5, 6, 23, 28 ). On day 1, K -to-Na ratio is significantly higher in ADX rats ( P = 0.005, by 2-way ANOVA) relative to sham rats. At day 2, this ratio plunged in ADX-WL rats, and also decreased in sham-WL, but more modestly. In sham-WL rats, starting on day 3, this ratio reversed and showed an upward slope of increase over the next 3 days. ADX-WL rats showed a sharper rise in this ratio between day 3 and 4. Although the urine K -to-Na ratio rose in the WL rats (both ADX and sham), after the initial period of natriuresis, it did not rise to a significantly higher level than that observed for the SD rats.
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Plasma biochemistry and body weight. Rats receiving the water load were hyposmotic and hyponatremic ( Table 1 ) and had significantly less weight gain. Plasma aldosterone and plasma corticosterone levels were increased significantly by the water load in sham-WL rats relative to sham-SD rats. No significant differences in these parameters were observed between ADX-WL and sham-WL, although there was a trend in plasma Na and osmolality to be lower in ADX-WL rats.

Table 1. Plasma physiology and body weight
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MAP. In Fig. 6, the daily treatment means of the MAP are plotted. As observed previously ( 29 ), water loading significantly increased the MAP ( Fig. 6 ) from day 3 ( P 0.028, by 2-way ANOVA). MAP rose slightly in all the four groups after they began the dDAVP infusion ( day -3 in Fig. 6 ). Divergence between the MAP means for SD- and WL-treated rats began at day 2 of the water loading from which point MAP for the water-loaded groups (sham-WL and ADX-WL) continued to rise. This increase was observed both in adrenalectomized and sham rats in this study ( Fig. 6 ), indicating that the escape-associated increase in MAP in this model was independent of changes in adrenal steroids. However, for sham-WL this rise was continued until day 5 after which it started coming down, while it continued to rise in ADX-WL group.* X! Q4 J2 s5 k
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Fig. 6. Mean arterial blood pressure ( n = 3/group). Water loading along with dDAVP significantly increased the MAP in both ADX and sham rats. dDAVP infusion ( day -3 ) also increased MAP by 10-15 mmHg in SD rats (and WL rats before initiating the water load). Tukey's multiple comparisons test was applied only when 1-way ANOVA detected a significant ( P / P9 `2 e! k* S+ ]9 X

ENaC subunits in whole kidney. Figure 7 shows the immunoblots obtained from whole kidney homogenates of the rats in this study, probed with antibodies against -, -, and -subunits of ENaC ( A, C, and E ) and their densitometric analysis ( B, D, and F ). Similar to our previous findings in cortical, outer medullary, and whole kidney ( 10, 29 ), we found an increase in -, -, and (both bands)-ENaC abundances with water loading, regardless of whether they were sham or ADX. In addition, the abundance of -ENaC was increased ( Fig. 7 A ) in ADX-SD relative to sham-SD, possibly due to slightly higher, but physiological level of plasma aldosterone in ADX-SD ( Table 1 ). Also, the increase in -ENaC with water load was most pronounced in the ADX rats ( Fig. 7, C and D ). The two bands associated with -ENaC were analyzed separately because they are known to be independently regulated ( 24 ). However, an increase of the 70-kDa band was not apparent between the ADX groups.
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Fig. 7. Immunoblotting of epithelial Na channel (ENaC) subunits in whole kidney. Left : each lane of each blot is loaded with a different rat's sample ( n = 5 for sham-SD, sham-WL, and ADX-SD and n = 6 for ADX-WL group) of whole kidney homogenate. Within each blot, equal amounts of total protein were loaded in each lane, and a Coomassie-stained gel confirmed equality of loading for each sample set. Blots were probed with polyclonal antibodies against -, -, and -epithelial Na channel (ENaC); A, C, and E, respectively. Right : summary of densitometry for -ENaC ( B ), -ENaC ( D ), and -ENaC ( F ) were presented. *Significant difference from sham-SD, from sham-WL, and from ADX-SD as determined by 1-way ANOVA followed by Tukey's multiple comparisons test.

Sodium cotransporters and water channels of CD: NCC. Figure 8, A and B, shows the immunoblots obtained from whole kidney homogenates of the rats in this study, probed with antibody against NCC and its densitometric analysis, respectively. As observed in previous studies ( 29 ), NCC was significantly increased by water loading in sham-WL rats relative to sham-SD ( Fig. 8, A and B ). In this study, the increase was also observed in the water-loaded adrenalectomized rats (ADX-WL) with clamped corticosteroid levels relative to ADX-SD ( Fig. 8, A and B ), indicating that the upregulation of NCC by water loading was at least partly independent of corticosteroid levels. However, final band density for the sham-WL rats was higher than that for the ADX-WL rats.( m" v! I: ^1 A, ^/ w, O

Fig. 8. Immunoblotting of other sodium cotransporters and water channels of collecting duct. Left : each lane of each blot is loaded with a different rat's sample ( n = 5 for sham-SD, sham-WL, and ADX-SD and n = 6 for ADX-WL group) of whole kidney homogenate. Within each blot, equal amounts of total protein were loaded in each lane, and a Coomassie-stained gel confirmed equality of loading for each sample set. Blots were probed with polyclonal antibodies against Na -Cl - cotransporter (NCC), aquaporin (AQP)-2, AQP-3, and Na -K -2Cl - cotransporter (NKCC2); A, C, E, and G, respectively. Right : summary of densitometry for NCC ( B ), AQP-2 ( D ), AQP-3 ( F ), and NKCC2 ( H ) was presented. *Significant difference from sham-SD, from sham-WL, and from ADX-SD as determined by 1-way ANOVA followed by Tukey's multiple comparisons test.+ `7 S- H5 d* j0 q: P9 w0 i
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AQP-2, -3, and NKCC2. As observed in previous studies ( 29 ), we found a significant decrease in AQP-2 abundance by water loading ( Fig. 8, C and D ). This decrease was also observed in the corticosteroid-clamped rats (ADX-WL; Fig. 8, C and D ). In addition, ADX rats had significantly increased AQP-2 expression compared with sham rats, by two-way ANOVA. In this study, no significant differences were observed for AQP-3 or NKCC2 among any of the four groups ( Figs. 8, E and F, and 7, G and H, respectively).

11 -HSD2. Figure 9 A shows a representative immunoblot loaded with whole kidney homogenates probed with antibody against 11 -HSD2. Below this is a densitometric bar graph summary. Similar to our previous findings ( 29 ), there were no significant differences in 11 -HSD2 protein abundance among the groups. However, ADX rats tended to have increased 11 - HSD2 protein abundance relative to sham rats ( P = 0.079 by 2-way ANOVA). In Fig. 9 B, the activities of 11 -HSD2 enzyme relative to sham-SD group were plotted for the groups, and there was no significant difference in the activity of this enzyme among the groups.
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Fig. 9. 11 HSD2 abundance and activity in whole kidney. A : immunoblot of 11 HSD2 and densitometry bar graph summary. Each lane is loaded with a different rat's sample ( n = 5 for sham-SD, sham-WL, and ADX-SD and n = 6 for ADX-WL group) of whole kidney homogenate. Equal amounts of total protein were loaded in each lane and a Coomassie-stained gel confirmed equality of loading. B : summary of relative 11 HSD2 activity as determined by the conversion of H 3 -labeled corticosterone to 11 dehydrocortisone.: h0 x# W% C, h# }  J' Q* p4 h
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DISCUSSION
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The aim of this study was to determine whether the rise in blood pressure and elevation of distal tubular sodium transporter and channels previously observed ( 10, 11, 29 ) during vasopressin escape were independent of the rise in aldosterone ( 29 ). The original data were correlative, and no cause and effect had been established. Therefore, in this study we adrenalectomized the rats and replaced the corticosteroids with clamped levels of aldosterone and corticosterone. In summary, we found that both the escape-associated rise in blood pressure as well as the upregulation of ENaC and NCC do not require an increase in plasma aldosterone, although some sensitivity to the increase in aldosterone was still apparent, especially for ENaC. In this study, we also found that vasopressin escape was associated with a significant increase in corticosterone, which was abolished by the clamp. This might also have an effect on escape and associated changes.

In general, we demonstrated that clamping of these hormone levels had little impact on the physiological characteristics of vasopressin-induced escape. We predicted that the adaptive sodium-retentive mechanism to prevent excessive sodium loss resulting from escape-associated natriuresis ( 29 ) would be attenuated in this model due to relatively lower aldosterone levels. In contrast, we found that the ADX-WL rats had a marginally blunted natriuresis with a recovery period that was similar, if not more defined, than that observed for the sham-WL rats, indicating that the aldosterone rise was not required to achieve sodium balance in these rats. This is evident by the sharp rise in the urine K -to-Na ratio in the ADX-WL rats after day 3, indicating restoration of sodium-retentive mechanisms after the initial natriuresis. In addition, there were no significant differences for plasma osmolality or plasma sodium concentration between these two groups. Nevertheless, there was a trend toward reduction in these two parameters in the ADX-WL rats. However, it is likely that we clamped the level of aldosterone in the ADX rats at a level which ended up being a bit higher than the sham-SD rats, based on plasma aldosterone levels and also based on our observation of reduced sodium excretion before the escape ( day 1 ) in ADX rats and increased K -to-Na ratio, both indicative of increased aldosterone activity. This may have preserved sodium balance somewhat in ADX-WL rats.2 j4 ]; O  Y4 s! N
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The diuresis, as determined by urine volume, was somewhat blunted in ADX-WL rats compared with sham-WL rats. Furthermore, significantly lower urine volume was observed in ADX groups relative to the sham groups at day 1. The mechanism underlying this blunting cannot be determined; however, it is consistent with significantly increased AQP-2 abundance (by 2-way ANOVA) in ADX rats relative to sham rats.) h8 e" P+ l( p/ n6 G( C" p

The clamped ADX rats had similar escape-associated elevated blood pressure as did sham-WL rats. In fact, in the sham-WL rats, blood pressure started to plateau or even decreased some after day 5, whereas in the ADX-WL rats it appeared to be continuing to rise. Nevertheless, it is clear from this study that a factor other than the relatively increased plasma aldosterone in the sham-WL rats (2.49 vs. 0.59 nmol/l in sham WL and ADX-WL, respectively) was responsible for the escape-associated increase in blood pressure. It is possible that it could be due to upregulated distal sodium transport.
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In fact, adrenalectomy with clamped corticosteroid levels had little effect on escape-associated changes in the protein abundances of sodium transporters, channel subunit, or that of AQP-2 water channel, for that matter. That is, we still saw a significant increase in the ADX-WL rats in NCC, -, -, and (both bands)-ENaC abundances relative to ADX-SD despite this clamp. However, the increase in NCC and -ENaC (70 kDa) was blunted some in the ADX-WL rats, suggesting some contribution from the markedly elevated aldosterone levels in the sham-WL rats.# a6 _- O* Z' _0 d+ P! W# L

Furthermore, the ENaC subunits appeared to be more sensitive, relative to NCC, to differences in circulating aldosterone levels in the low physiological range, i.e., below 1 nmol/l. That is, we saw a relative increase in -ENaC and the 70-kDa band of -ENaC in the ADX-SD rats relative to the sham-SD rats. Measured plasma aldosterone levels for these two groups were 0.016 and 0.67 nmol/l, respectively. However, we did not see any difference between these two groups for NCC abundance.

However, the ADX-WL and ADX-SD had very similar final plasma aldosterone levels and despite this, the pattern of protein changes in the WL rats resembled an "aldosterone-like pattern," with increased -ENaC, 70-kDa band of -ENaC, and NCC. Thus it is possible that these effects are due to some other steroid or steroid-like hormone released during vasopressin escape that has activity at the mineralocorticoid receptor, for example, corticosterone. It has been shown that the physiological effects of inhibiting 11 -HSD2 activity by carbenoxolone on distal tubular function in adrenalectomized rats (with clamped corticosteroid levels) were similar to those by intravenous infusion of high-dose aldosterone ( 3 ). However, here, and in our previous study, we saw no significant difference in 11 -HSD2 protein abundance or cellular expression pattern in escape ( 29 ). Previously, we did observe an increase with dDAVP infusion, confirming the results of Brooks et al. ( 4 ) showing that vasopressin infusion increases 11 -HSD2 gene expression. Moreover, 11 -HSD2 activity was not decreased in WL rats of either sham or ADX groups, which might have also explained this pattern of sodium transporter and channel subunit changes independent of changes in 11 -HSD2 protein levels. On the other hand, it is also possible that the regulation of these proteins is completely independent of the mineralocorticoid receptor.# b3 s, D9 B, j! k2 f' C; e
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For example, ANG II has been shown to increase -ENaC abundance in rats ( 2 ). However, we suspect that there is not an increase in circulating levels of ANG II because renin activity is about 25-fold decreased in this model ( 29 ). However, there is certainly a possibility of increased activity of the local renal renin-angiotensin-aldosterone system (RAAS) or in angiotensin-converting enzyme (ACE) activity and/or ANG II receptor (AT 1 ) regulation. However, Beutler et al. ( 2 ) did not show an increase in NCC abundance with ANG II, which suggests that ANG II is not likely to be the entire story.

Furthermore, despite the relatively increased abundance of NCC and -ENaC in the WL rats (both sham and ADX), we were unable to show an increase in the urinary K -to-Na ratio or a decrease in absolute sodium excretion, relative to the SD rats. This may suggest that the pressure-natriuresis clearly evident in the early phases of vasopressin escape is still occurring in the later phases likely driven by the persistently elevated blood pressure. Moreover, when you consider lower food intake in the WL rats during at least some periods of the escape, net sodium balance in the WL rats would be predicted to be lower in these rats, relative to SD. However, net sodium balance may have been reduced even more so had there not been an increase in these distal tubular sodium reabsorptive pathways.

Overall, it is clear that adrenalectomy plus clamping of the levels of circulating corticosteroids during vasopressin escape, which are normally increased in this model, does not abolish the rise in ENaC subunit or NCC abundances nor attenuate the increase in blood pressure observed. Thus an alternative to aldosterone must be considered. In addition, we provide evidence for potential differences in sensitivity of ENaC vs. NCC in its response to changes in aldosterone levels, i.e., in what range they occur. Finally, whether the link between the rise in sodium transporter and channel expression and blood pressure in this model is causal has not been established. Further studies are required, although increased activity of NCC and ENaC has been linked to hypertension in Gordon's syndrome and Liddle's syndrome, respectively, indicating that this idea is plausible.6 f" @6 y, A: }( A- v7 N
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GRANTS

This work was primarily supported by National Heart, Lung, and Blood Institute Grant HL-074142 (to C. Ecelbarger). Also, Grants HL-073193 and DK-064872 supplied additional salary support for C. Ecelbarger, S. Tiwari, and X. Hu. Finally, a Research Award from the American Diabetes Association helped support C. Ecelbarger and Y. Sugimura.# c& @' O) Q9 r
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ACKNOWLEDGMENTS0 n$ I% @  k. B: p4 D

We thank J. Song for preliminary work and V. Kumar for assistance with plasma analysis.: P) @! r! P7 Q& M8 D4 x3 i
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