Collecting duct-specific knockout of the endothelin A receptor alters renal vaso

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Division of Nephrology, University of Utah Health Sciences Center, Salt Lake City
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+ @% C8 d$ s* K4 u7 WSalt Lake Veterans Affairs Medical Center, Salt Lake City, Utah9 G" B' r* `! G% N: z* f1 G; M

+ r2 H1 V$ ?; b/ o- dHoward Hughes Medical Institute, University of Texas Southwestern, Dallas, Texas; E3 C8 F; F: r9 h  B- S! d

( M) q6 o6 x6 m1 L; ?4 t; p3 u' Z0 DABSTRACT4 N  \( X. V3 h: m& \+ k# V( E

0 I5 l! Y  z0 S" W& \# c# J  xCollecting duct (CD)-specific knockout (KO) of endothelin-1 (ET-1) causes hypertension, impaired ability to excrete a Na load, and enhanced CD sensitivity to the hydrosmotic effects of vasopressin (AVP). CD express the two known ET receptors, ETA and ETB; in the current study, the role of the CD ETA receptor in mediating ET-1 actions on this nephron segment was evaluated. The ETA receptor gene was selectively disrupted in CD (CD ETA KO). CD ETA KO mice had no differences in systemic blood pressure, Na or K excretion, and plasma aldosterone or renin activity in response to a normal- or a high-Na diet compared with controls. During normal water intake, urinary osmolality (Uosm), plasma Na concentration, and plasma osmolality were not affected, but plasma AVP concentration was increased in CD ETA KO animals (0.57 ± 0.25 pg/ml in controls and 1.30 ± 0.29 pg/ml in CD ETA KO mice). CD ETA KO mice had a modestly enhanced ability to excrete an acute, but not a chronic, water load. DDAVP infusion increased Uosm similarly; however, CD ETA KO mice had a more rapid subsequent fall in Uosm during sustained DDAVP administration. CD suspensions from CD ETA KO mice had a 30–40% reduction in AVP- and forskolin-stimulated cAMP accumulation. These data indicate that CD ETA KO decreases renal sensitivity to the urinary concentrating effects of AVP and suggest that activation of the ETA receptor downregulates ET-1 inhibition of AVP actions in the CD. Furthermore, the CD ETA receptor does not appear to be involved in modulation of systemic blood pressure or renal Na excretion under physiological conditions.6 H# [# I. E! W. I
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tubule; adenosine 3', 5'-cyclic monophosphate; inner medullary collecting duct
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, f0 G8 ?' W6 h0 lRECENT STUDIES INDICATE THAT collecting duct (CD)-derived endothelin-1 (ET-1) regulates systemic blood pressure (BP) and renal Na and water excretion. The CD is the major renal site of ET-1 synthesis (3, 13, 24, 30, 31) and binds relatively large amounts of the peptide (4, 26, 27). In vitro studies noted that exogenous ET-1 inhibits mineralocorticoid and AVP-stimulated Na and Cl reabsorption in isolated cortical CD (CCD) (16, 18, 29), decreases Na-K-ATPase activity in suspensions of inner medullary CD (IMCD) (34), reduces AVP-stimulated water permeability in CCD (26, 29), and inhibits AVP-stimulated cAMP accumulation (6, 15, 28) and osmotic water permeability (20, 22) in IMCD. We recently reported that CD-specific knockout (KO) of the ET-1 gene causes hypertension and impaired ability to excrete a Na load (1). In a separate study, CD-specific KO of the ET-1 gene caused enhanced AVP responsiveness, as evidenced by reduced plasma AVP concentration, increased urine osmolality in response to DDAVP, impaired ability to excrete an acute water load, and elevated IMCD cAMP accumulation in response to AVP or forskolin (9). Collectively, these data indicate that CD-derived ET-1 is an important physiological regulator of systemic BP and renal salt and water excretion.
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The mechanisms by which CD-derived ET-1 exerts these effects are incompletely understood. Most studies have addressed the potential for ET-1 functioning in an autocrine manner in the CD. Numerous studies have shown that CDs express ETB receptors (4, 11, 14, 26, 27). Exogenous ET-1 inhibition of AVP actions in vitro is mediated through activation of the ETB receptor (6, 15, 26, 29). There is controversy, however, over the expression and biological significance of the ETA receptor in the CD. Studies using autoradiography of whole kidneys or RT-PCR of dissected nephron segments detected only ETRB in the CD of rats or humans (4, 10, 26, 27). In contrast, immunohistochemical binding and RT-PCR analysis have detected ETA receptor expression by acutely isolated or cultured CD from rats or dogs (2, 6, 14, 23, 32, 33). Furthermore, ETA receptor activation reduces endothelial nitric oxide (NO) synthase expression in dog IMCD (33), while higher concentrations of ET-1 increase Na channel activity via activation of the ETA receptor in A6 cells, a distal nephron cell line (7). The reasons for the above discordant results may derive, at least in part, from the relative scarcity of ETA receptors in the CD. For example, the ratio of ETB:ETA receptors may exceed 4:1 in CD (6). Thus, while the CD ETB receptor seems to mediate much of the inhibitory effect of ET-1 on Na and water reabsorption, at least in vitro, the role of the CD ETA receptor is very much an open question.3 M, w5 f) Z7 [8 N/ z

; ~1 Y5 C) O6 b( M& @The current study examined the role of the CD ETA receptor in regulating systemic BP and renal Na and water excretion. ETA receptors are widely expressed in the vasculature, so systemic administration of ETA receptor antagonists or agonists would potentially affect renal hemodynamics and systemic BP, independent of any effects on the CD. ETA receptor KO mice die at birth with severe craniofacial deformities and defects in the cardiovascular system (5), making them unfeasible for study. Consequently, the current study employed the Cre/lox system to selectively delete the ETA receptor in CD. We now report that ETA receptor KO mice have altered AVP responsiveness, but no differences in systemic BP or renal Na excretion. These studies provide the first direct in vivo evidence for a physiological role of CD ETA receptors in regulating renal function.
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3 S7 C7 g) B* I4 T: c' OMETHODS' O1 W. S% i; I$ u

8 b# H$ J$ J5 STransgenic mice lines. Mice with CD-specific disruption of the ETA receptor gene were generated in a manner similar to that previously described (1). Briefly, mice containing the loxP-flanked (floxed) ETA receptor gene (obtained from Dr. M. Yanagisawa at the Howard Hughes Institute at University of Texas Southwestern Medical Center) were mated with aquaporin-Z (AQP2)-Cre mice. The floxed mice contain exons 6–8 of the ETA receptor gene flanked by loxP sites [these exons are critical to ETA receptor gene functional expression (5)]. AQP2-Cre mice contain a transgene with 11 kb of the mouse AQP2 gene 5'-flanking region driving expression of Cre recombinase. These mice are phenotypically identical to those previously described by our group in which 14 kb of the human AQP2 promoter were used to drive Cre expression (21). An SV40 nuclear localization signal is located on the NH2 terminus of Cre and an 11-amino acid epitope tag, derived from herpes simplex virus glycoprotein D, is located on the COOH terminus of Cre. Female AQP2-Cre mice were mated with male floxed ETA receptor mice; female offspring heterozygous for both AQP2-Cre and floxed ETA receptor were bred with males homozygous for floxed ETA receptor. Animals homozygous for floxed ETA receptor and heterozygous for AQP2-Cre (CD ETA KO) were used in all studies. Sex-matched littermates that were homozygous for the floxed ETA receptor gene, but without Cre, were used as controls in all studies.
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Genotyping. Tail DNA was prepared by standard methods and PCR was amplified for the AQP2-Cre transgene using oligonucleotide primers mAQP2 F (5'-CCT CTG CAG GAA CTG GTG CTG G-3') and CreTag R (5'-GCG AAC ATC TTC AGG TTC TGC GG-3'), which amplify the 671-bp junction between the mouse AQP2 promoter and the Cre gene (1). The ETA receptor gene was amplified using RAF102F2 5'-CCC ATG CTT AGA CAC AAC CAT G-3' and ETA genoR2 5'-GAT GAC AAC CAA GCA GAA GAC AG-3'. These primers are able to distinguish zygosity of the animals by spanning the loxP site upstream of exon 6. The wild-type allele product is 314 bp and floxed allele is 354 bp. PCR products were visualized after electrophoresis through 1.5% agarose.
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Metabolic balance studies. All mice were studied at 2–3 mo of age. All mice were acclimated for 1 wk to Nalgene (Rochester, NY) metabolic cages. Mice were fed 9 ml of a gelled diet that contained all nutrients and water as previously described (1). BP and pulse were determined daily by tail-cuff plethysmography (BP-2000, Visitech Systems, Apex, NC) for 1 wk. The hemodynamic values were not recorded during this conditioning period. After 1 wk, metabolic balance studies were performed for 3 consecutive days. Daily gel intake and body weights were measured, and urine was collected under oil. BP and pulse were determined daily and were averaged from 8–10 recordings each day. Mice were then killed by guillotine, and blood was collected for plasma Na, osmolality, and AVP determination.
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For sodium-loading studies, mice were studied as described above except that after 3 days of a normal (0.3%)-sodium diet, 20 μl of blood were removed by tail vein stick for determination of plasma renin activity (PRA). Mice were then placed on a high-sodium diet for 10 days. The high-sodium diet consisted of 11 ml of gelled diet containing 2% sodium. Daily weights and vital signs were obtained, and urine was collected. At the end of this period, mice were tail-bled for PRA and plasma renin concentration determination.
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( j* q# B) L, A- ~$ K0 _) _5 |For chronic water loading, mice were acclimatized to metabolic cages and a normal water-gelled diet for 1 wk, balance studies as described above were performed for 3 more days, then the animals were switched to a high-water diet for up to 7 days. The high-water diet consisted of 19 ml of gelled diet containing 13.4 ml water and the same amount of nutrients and electrolytes as the normal water diet. Daily gel intake was measured and urine was collected. After 7 days, mice were killed by guillotine and blood was collected for plasma Na, osmolality, and AVP determination.! v* U3 @4 K, q8 U7 p3 I

. m( ?; _! o8 Z+ y! \$ ?! ~DDAVP studies. Mice were acclimatized to metabolic cages, and 3 days of baseline measurements were taken as described above. Subsequently, mice were given DDAVP (0.25 ng/h, Sigma, St. Louis, MO) via subcutaneous osmotic minipump (Alzet model 1002, Alzet, Cupertino, CA) for up to 7 days, while being on a normal water intake (9 ml of the normal water intake gelled diet). For combined DDAVP and water loading, mice were studied as above until day 4 of DDAVP, then switched to the high-water diet (containing 19 ml of gelled diet) and maintained on this diet and DDAVP for up to 7 days. At the conclusion of the DDAVP ± water loading studies, mice were killed by halothane inhalation, and blood was obtained for electrolyte determination.
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Acute water loading. Mice were acclimatized to metabolic cages and a gelled normal water diet for 1 wk. They were then placed into small metabolic cages that contained no food or water and given 1.5 ml of water intraperitoneally. Subsequently, urine was collected under oil on an hourly basis for the next 6 h. Urine was analyzed for volume and osmolality.
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cAMP studies. IMCD were acutely isolated in a manner similar to that previously described (15). Briefly, inner medulla were minced in Kreb's buffer with 1 mg/ml collagenase (Type IV, Worthington, Lakewood, NJ) and 0.1 mg/ml DNase (Sigma) and incubated at 37°C for 30–45 min. IMCD fragments were washed and all subsequent incubations were done in Kreb's buffer. IMCD were incubated in 1 mM isobutylmethylxanthine (Sigma) for 30 min before addition of varying concentrations of AVP or 1 μM forskolin for 10 min. Cells were extracted with ethanol, and cAMP was measured by ELISA (R&D Systems). Total cell protein was measured by the Bradford assay (Bio-Rad, Hercules, CA).! F  R( E5 I8 e6 I
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ETA receptor mRNA expression. Kidney sections were incubated in 1 mg/ml collagenase at 37°C for 1 h. CCD and IMCD were dissected, and RNA was isolated as previously described (19). Samples were reverse transcribed and real-time PCR was performed using a Smart Cycler (Cephid, Sunnyvale, CA). The primer sequences for ETA receptor mRNA were EdnrA F2 5'-CCT GCC TCT GTT GCT GTT GT-3' and EdnrA R2 5'-CGT TCC GTG TTG TGG TTG TT-3', which yield a product size of 112 bp. The primers for GAPDH were GAPDH F 5'-TGG CCT CCA AGG AGT AAG AA-3' and GAPDH R 5'-CTG GGA TGG AAA TTG TGA GG-3', which yield a product size of 110 bp. The ratio of ETRA receptor to GAPDH mRNA was calculated for each sample.
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Electrolyte and hormone analysis. Plasma and urine were analyzed for Na and K concentration (EasyVet analyzer, Medica, Bedford, MA), osmolality (Osmett II, Precision Systems, Natick, MA), and creatinine (Jaffé colorimetry, Sigma). For AVP determination, heparinized plasma was extracted with acetone and petroleum ether (9). AVP was assayed using a radioimmunoassay kit (Peninsula Laboratories, San Carlos, CA). PRA was measured as previously described (1) using an indirect radioimmunoassay (Peninsula Laboratories). Plasma renin concentration was determined as for PRA, with the exception that excess angiotensinogen was added. Urine from basal and sodium loading studies was analyzed for 24-h aldosterone excretion. Urine was hydrolyzed with HCl, ethyl acetate was extracted, and aldosterone was determined by radioimmunoassay (Coat-a-Count, Diagnostic Products, Los Angeles, CA).( i, |6 ~* W: M7 i* h. F
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Statistics and ethics. Comparisons between floxed ET-1 and CD ETA KO mice were analyzed by the unpaired Student's t-test. Comparisons of multiple points (urine volume, urine osmolality, BP, sodium excretion, cAMP accumulation) were made using one-way ANOVA with the Bonferroni correction. P
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RESULTS
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Characterization of CD ETA KO mice. CD ETA KO mice developed normally until at least 1 yr of age and had no gross morphological abnormalities. As previously described (1), AQP2-Cre mice confer CD-specific knockout, as determined by principal cell-specific Cre recombinase activity in mice heterozygous for AQP2-Cre and the ROSA26-YFP reporter, in situ hybridization, genomic PCR of microdissected CD, and genomic PCR for gene recombination in an organ panel of 15 different organs. This was further confirmed by real-time PCR of ETA receptor mRNA in microdissected CD. As expected, CD from CD ETA KO mice had virtually no detectable ETA receptor mRNA (2% of the levels expressed in control CD, n = 30 tubules). Thus CD ETA KO mice have CD-specific inactivation of the ETA receptor gene.
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( R9 \7 U5 F0 ^4 j- r  j6 LRenal function and systolic BP during a normal- or a high-Na diet. As previously described (1), all mice were ration fed so they had exactly matched food and water intake. This was achieved by using a gelled diet that contained all food and water and that met all nutritional needs. Mice eat all of the provided gel (9 ml of gel containing 6.3 ml of water) and do not drop any of it into the bottom of the cage (it is quite gummy). Under these baseline conditions, there were no differences in systolic BP, pulse, body weight, urine volume, urine Na or K concentration, plasma Na or K concentration, or urine Na or K excretion (Table 1 and Figs. 1 and 2). After being given a high-Na diet, in which urine Na excretion increased fivefold, there remained no differences between control and CD ETA KO mice with respect to systolic BP, pulse, urine volume, urine Na or K concentration, or urine Na or K excretion for up to 10 days of Na loading (Table 2 and Figs. 1 and 2). Over the initial 2 days of Na loading, body weight increased by 0.5 ± 0.2 g in control mice and 0.6 ± 0.2 g in CD ETA KO mice, while after 1 wk of Na loading both groups increased in body weight by 1.0 ± 0.3 g. Urinary aldosterone excretion and PRA did not differ between CD ETA KO and control mice on a normal-Na diet (Table 3). Both urinary aldosterone excretion and PRA fell after Na loading, but this was to the same degree in both groups of mice (Table 3). There was no difference in plasma renin concentration between CD ETA KO and control mice on a high-Na diet (Table 3).0 t- W% t8 F/ o$ G

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Renal function during normal and high water intake. Mice were fed the same baseline normal-Na diet as described above. This resulted in no differences between CD ETA KO and control mice with respect to urine volume or plasma Na concentration (as described above) or urine or plasma osmolality (Table 4). However, plasma AVP concentration was significantly greater in CD ETA KO mice, despite all other aspects of water metabolism being similar (Table 4). These data indicate that CD ET-1 KO mice excrete similar amounts of water and osmolytes compared with control mice but that this occurs at higher plasma AVP levels.% w0 ]$ l" v' u

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6 `0 c! y' T( {4 }" nCD ETA KO and control mice were chronically water loaded, as evidenced by a four- to fivefold increase in urine volume and a threefold decrease in urine osmolality (Fig. 3). Mice ate all the gel and had precisely matched fluid intake (13.4 ± 0.2 ml in controls and 13.3 ± 0.2 ml in CD ETA KO). As shown in Fig. 3, there was no difference in urine volume or urine osmolality between CD ETA KO and control mice for up to 6 days of increased water intake. Similarly, after 6 days of water loading, there was no difference in plasma Na concentration or plasma osmolality between the two groups (data not shown), whereas plasma AVP levels were suppressed to undetectably low levels. Over the initial 2 days of water loading, body weight increased by 0.6 ± 0.2 g in control mice and 0.7 ± 0.2 g in CD ETA KO mice, while after 6 days of water loading, body weight increased by 1.1 ± 0.3 g in controls and 1.0 ± 0.2 g in CD ETA KO mice. Thus CD ETA KO mice have no apparent defect in their ability to handle a chronic water load.
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/ @: p: U; s" N, ~Renal function during DDAVP administration. The increased plasma AVP levels during a normal-water diet in CD ETA KO mice suggested that these mice have reduced responsiveness to the hydrosmotic effects of AVP. To test this, mice were given DDAVP for 3 days via osmotic minipump, while being on a normal water intake (9 ml of the normal water intake gelled diet). This procedure was designed to fix plasma AVP at maximal water-retaining levels. After 1 day of DDAVP administration, urine osmolality increased, and urine volume decreased in CD ETA KO mice and control mice; the magnitude of these changes was similar between the two groups (Fig. 4). Of note, after 2 days of DDAVP administration, CD ETA KO mice had a greater fall in urine osmolality compared with controls (Fig. 4). No differences in urine volume were seen between the two groups; however, the predicted small difference in urine volume (based on the difference in urine osmolality) would be undetectable given the inherent high variability in urine volumes within groups. Plasma Na or osmolality was not determined at this time point as it would result in too large a blood loss and potentially confound data obtained on subsequent days. However, after 3 days of DDAVP, there was no difference in either plasma Na concentration or plasma osmolality between the two groups of mice (data not shown). Weight gain over the 3 days of DDAVP was similar between the two groups of mice (1.5 ± 0.4 g in CD ETA KO and 1.7 ± 0.3 g in controls). Thus these data further suggested that CD ETA KO mice may have reduced responsiveness to AVP.
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To determine whether CD ETA KO affects AVP "escape" during water loading, mice were given concurrent DDAVP and high water intake. As shown in Fig. 5, there was again a significantly greater drop in urine osmolality on day 2 of DDAVP administration during normal water intake. Urine volume tended to be greater in CD ETA KO mice at this time point, although it did not achieve statistical significance. Subsequent water loading caused a similar drop in urine osmolality and an increase in urine volume in CD ETA KO and control mice; there was no difference in these parameters on any day during the 4 days of water loading during DDAVP administration. There was also no significant difference in plasma Na concentration or plasma osmolality after 4 days of DDAVP and water loading (data not shown). CD ETA KO and control mice gained similar amounts of weight over 4 days of DDAVP and water loading (1.1 ± 0.3 g in CD ETA KO and 1.0 ± 0.3 g in controls). Thus CD ETA KO does not affect the ability to escape from the hydroosmotic effects of DDAVP and high water intake.
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Effect of acute water loading. Although CD ETA KO mice exhibited decreased AVP responsiveness, no data indicated that this was of significant physiological relevance. A problem with these studies was that chronic water loading studies measured renal function only at 24-h intervals and not until 24 h after water loading. It was possible, therefore, that differences in renal water handling might be observed more acutely. Consequently, mice were given 1.5 ml ip of water, followed by hourly urine collections for 6 h. As shown in Fig. 6, CD ETA KO mice had a more rapid drop in urine osmolality and then began to normalize urine osmolality sooner than controls. As for the above studies with DDAVP, although statistically significant, these were rather modest differences. Notably, no differences in urine volume could be detected after acute water loading (however, as stated previously, urine volumes are normally highly variable within groups). Nonetheless, these data suggest that CD ETA receptors may play a role in modifying the diuretic response to water loading, at least within the first several hours of increased water intake.
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/ P: f3 k$ s+ e* O, TAgonist-stimulated cAMP accumulation. To more directly assess increased CD sensitivity to AVP in CD ETA KO mice, IMCD were acutely isolated and stimulated with AVP. As previously noted (9), outer MCD or CCD cannot be studied due to the inability to acutely isolate sufficient quantities of these nephron segments. IMCD from CD ETA KO kidneys had decreased AVP-stimulated cAMP accumulation, and this occurred at AVP concentrations as low as 100 pM (Fig. 7). To determine whether this augmented AVP response was due, at least in part, to postreceptor mechanisms, IMCD were stimulated with forskolin. Similar to AVP, forskolin increased cAMP levels less in IMCD from CD ETA KO mice compared with controls (Fig. 7). Thus at least part of the reduced AVP responsiveness observed in vivo can be related to decreased AVP-induced CD cAMP accumulation via postreceptor mechanisms.
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The present study demonstrates that the CD ETA receptor regulates renal AVP responsiveness. In contrast, CD-specific knockout of the ETA receptor did not affect BP or urinary Na excretion in mice on either a normal- or a high-Na diet. CD-specific knockout of the ETA receptor increased plasma AVP levels yet did not change renal water excretion under baseline conditions or during chronic water loading. Hence, although the relationship between plasma AVP levels and urine osmolality was reset, other mechanisms were fully compensated. When plasma AVP levels were fixed at superphysiological levels by exogenous AVP administration, CD ETA KO mice exhibited a more rapid decrease in urine osmolality after the initial exogenous AVP-induced increase in urine concentration. Furthermore, CD ETA KO mice had a more rapid decrease in urine osmolality following an acute water load. Although statistically significant, these alterations in urine osmolality in CD ETA KO mice were not accompanied by an observable difference in urine volume (as stated earlier, the failure to detect differences in urine volume may relate, at least in part, to the inherently high variability in urine volumes). Importantly, the decreased AVP responsiveness in CD ETA KO mice was confirmed by demonstrating decreased AVP-induced cAMP accumulation in acutely isolated CD ETA KO IMCD. Taken together, these studies indicate that deficiency of the CD ETA receptor causes reduced AVP sensitivity, associated with decreased urine concentration at a given circulating AVP level.
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The lower AVP responsiveness in CD ETA KO mice is in contrast to the elevated AVP sensitivity, and impaired ability to excrete an acute water load, in CD ET-1 KO mice (9). The CD ET-1 KO studies indicated that CD-derived ET-1 functions as an inhibitor of AVP action, facilitating a diuresis. The current study indicates, therefore, that activation of the CD ETA receptor opposes the predominantly diuretic effect of ET-1. This implies that ET-1 must exert its diuretic actions through either activation of the CD ETB receptor and/or through paracrine effects. The former possibility is likely involved, at least to some degree; in vitro studies have demonstrated that activation of the CD ETB receptor causes inhibition of AVP-stimulated cAMP accumulation and osmotic water permeability (6, 15, 28). Hence, the current study suggests that CD ETA and ETB receptors have opposing modulatory effects on AVP-induced signaling. Because the predominant effect of CD-derived ET-1 is to facilitate a diuresis, the CD ETB receptor must be preferentially activated by ET-1. Why and when CD ETA receptors are activated remain speculative; however, certain possibilities are suggested by previous investigations. In particular, one group has reported differential activation of ETA and ETB receptors in A6 cells, a distal nephron cell line, depending on the concentration of ET-1 (7). Low concentrations of ET-1 activate the ETB receptor with resultant inhibition of Na transport, while substantially higher concentrations of ET-1 activate the ETA receptor and stimulate Na transport (7). If this system is analogous to that seen in CD, then CD ETA receptor activation might only occur under conditions in which CD ET-1 production is very high. One such circumstance would be during acute water loading where CD ET-1 production has been shown to be greatly increased (9). In this case, activation of the ETA receptor may serve to brake the diuretic effects of ET-1. Many such counterregulatory biological systems are known; they presumably exist to avoid an unbalanced effect of any given regulatory factor. Clearly, further studies are needed to characterize the specific conditions under which the CD ETA receptor is biologically relevant. In particular, it will be interesting to see the role of these receptors in diseases of the kidney where renal ET-1 levels are markedly elevated (12).
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The mechanisms by which CD ETA receptor deficiency leads to reduced AVP-stimulated cAMP accumulation in IMCD are unknown. The finding that CD ETA receptor deficiency also causes decreased forskolin-stimulated cAMP accumulation in acutely isolated IMCD indicates that the lower AVP responsiveness is due, at least partially, to postreceptor mechanisms. There is no information on signaling pathways that are activated by the ETA receptor in CD. However, based on findings in vascular smooth muscle cells, one interesting possibility is suggested. ET-1, via ETA receptor activation, increases superoxide levels in the vasculature and in the urine (17, 25). Such an increase in CD superoxide production, if it did indeed occur, might lead to reduced NO levels. ET-1 is well known to stimulate NO production through activation of the ETB receptor, and NO has been demonstrated to reduce AVP-stimulated water permeability in isolated CCD (8). Thus the potential exists for ETA receptor-stimulated superoxide production to reduce ETB receptor-induced NO levels, thereby mitigating the inhibitory effect of ET-1 on AVP action. Key studies needed to investigate this scenario include determination of whether ET-1 does, in fact, inhibit AVP action in the CD through NO, whether CD ETA receptor activation increases superoxide formation, and whether such superoxide formation reduces CD NO levels.8 _# h5 \) ~; ]2 `

4 g1 ~$ H! U6 p% p3 z1 F: pThe finding that CD ETA KO mice have no changes in systolic BP or renal Na excretion is in contrast to studies wherein CD ET-1 KO caused hypertension and impaired Na excretion (1). This indicates that the CD ETA receptor does not play a role in mediating CD-derived ET-1 effects on salt transport. This further suggests that the signaling systems activated by ET-1 that regulate Na vs. water excretion are, at least to some extent, different. The nature of such signaling systems, as discussed above, is an important area for further investigation.! I- i. _4 p* ~4 X& P9 E
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In summary, the present studies demonstrate that activation of the CD ETA receptor, under physiological circumstances, opposes the predominant diuretic effect of ET-1. This most likely occurs through increased sensitivity to AVP-induced cAMP accumulation. The CD ETA receptor does not appear to be involved in physiological regulation of systemic BP or renal Na excretion. Notably, these studies clearly demonstrate that the CD does, in fact, express ETA receptors and that they have physiological relevance.6 h# a6 O% l( `, R
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GRANTS
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This research was funded in part by National Institutes of Health Grants DK-59047 and DK-96392 (to D. E. Kohan).0 B) r1 x" `# f0 e2 l1 b

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There is no conflict of interest for these studies.
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ACKNOWLEDGMENTS1 k: E/ z1 g- P" Y
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The technical assistance of Dr. L. Miller, Dr. A. Rohrwasser, and E. Hillas (latter two in Dr. J.-M. Lalouel's laboratory) is appreciated.
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FOOTNOTES+ i+ K! p( G1 I: M& E
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.. n( ]1 u7 n" j/ f# |

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大小年

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