Collecting duct-specific knockout of endothelin-1 alters vasopressin regulation of urine osmolality
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《美国生理学杂志》
Division of Nephrology, University of Utah Health Sciences Center, Salt Lake City, Utah
Department of Physiology, Kosin University College of Medicine, Kosin, South Korea
Howard Hughes Medical Institute, University of Texas Southwestern, Dallas, Texas
Division of Endocrinology and Metabolism, Georgetown University, Washington, District of Columbia
Salt Lake Veterans Affairs Medical Center, Salt Lake City, Utah
ABSTRACT
In vitro studies suggest that endothelin-1 (ET-1) inhibits vasopressin (AVP)-stimulated water permeability in the collecting duct (CD). To evaluate the role of CD-derived ET-1 in regulating renal water metabolism, the ET-1 gene was selectively disrupted in the CD (CD ET-1 KO). During normal water intake, urinary osmolality (Uosm), plasma Na concentration, urine volume, and renal aquaporin-2 (AQP2) levels were unchanged, but plasma AVP concentration was reduced in CD ET-1 KO animals. CD ET-1 KO mice had impaired ability to excrete an acute, but not a chronic, water load, and this was associated with increased CD ET-1 mRNA in control, but not CD ET-1 KO, mice. In response to continuous infusion of 1-desamino-8-D-arginine vasopressin, CD ET-1 KO mice had greater increases in Uosm, V2 and AQP2 mRNA, and phosphorylation of AQP2. CD suspensions from CD ET-1 KO mice had enhanced AVP- and forskolin-stimulated cAMP accumulation. These data indicate that CD ET-1 KO increases renal sensitivity to the urinary concentrating effects of AVP and suggest that ET-1 functions as a physiological autocrine regulator of AVP action in the CD.
aquaporin-2; adenosine 3',5'-cyclic monophosphate; inner medullary collecting duct
IN VITRO STUDIES SUGGEST THAT collecting duct (CD)-derived endothelin-1 (ET-1) may be an autocrine inhibitor of vasopressin (AVP)-stimulated water reabsorption in the kidney. Within the kidney, the CD is the major renal site of ET-1 synthesis (6, 18, 31, 41, 42) and possibly binding (7, 35, 36). In vitro, exogenous ET-1, through activation of the ETB receptor (ETRB), reduces AVP-stimulated water permeability in cortical CD (35, 40) and inhibits AVP-stimulated cAMP accumulation (10, 21, 39) and osmotic water permeability (27, 29) in inner medullary CD (IMCD). Blockade of endogenous ET-1 action increases AVP-stimulated cAMP levels in cultured IMCD cells (19), providing the most direct evidence for a potential autocrine role of the peptide.
There is less convincing, and no direct, evidence demonstrating an autocrine role for ET-1 in regulating CD water transport in vivo. Part of the problem stems from the inability to discriminate between direct ET-1 effects on the nephron as opposed to the vasculature. Administration of ET agonists or antagonists usually alters renal plasma flow and glomerular filtration rate, effects that can impact urinary water excretion. Studies have shown that (17) systemically administered ET, at doses with minimal detectable effects on renal hemodynamics, increases water excretion (14, 16, 32). Additionally, chronic water loading in rats and humans is associated with elevated urinary ET-1 excretion (although the source of urinary ET-1 could not be fully ascertained) (20, 24, 46), whereas water deprivation decreases urinary ET-1 excretion (20, 25). Thus circumstantial evidence suggests that ET-1 may inhibit renal water reabsorption. However, the physiological role that the peptide plays in regulating renal water transport, particularly through autocrine regulation of CD function, remains unknown.
Alternative methods for examining the physiological role of CD-derived ET-1 in regulating renal water excretion have not yet been employed. Traditional gene knockout methodologies have proven problematic in that every mouse homozygous for knockout of a component of the ET system [ET-1, ET-2, ET-3, ET receptor A (ETRA), ETRB] has a lethal phenotype (2, 15, 22, 23, 30). Renal function has been assessed on a limited basis in spotted lethal rats, which carry a natural mutation in the ETRB gene resulting in toxic megacolon (13). Spotted lethal rats that have been "rescued" with a transgene expressing ETRB under control of the dopamine -hydroxylase promoter exhibit hypertension; however, renal water excretion has not been reported in this model. Furthermore, these animals lack ETRB expression in the kidney [almost every cell in the kidney expresses ETRB (17)], while circulating ET-1 levels are likely to be markedly elevated (3). Consequently, this model was not employed. We recently reported the development of mice with CD-specific knockout of ET-1. These mice express Cre recombinase under control of the aquaporin-2 (AQP2) promoter and are homozygous for loxP-flanked exon 2 of the ET-1 gene. We now report that these mice have altered renal water handling, thereby providing direct evidence for a physiological role of CD-derived ET-1 in regulating renal water excretion.
METHODS
Transgenic mice lines. Mice with CD-specific disruption of the ET-1 gene were generated in a manner similar to that previously described (1). Briefly, mice containing the loxP-flanked (floxed) ET-1 gene (obtained from Dr. M. Yanagisawa at the Howard Hughes Institute at University of Texas Southwestern Medical Center) were mated with AQP2-Cre mice. The floxed mice contain exon 2 of the ET-1 gene flanked by loxP sites [exon 2 is critical to ET-1 gene functional expression (23)]. 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 (28). 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 ET-1 mice; female offspring heterozygous for both AQP2-Cre and floxed ET-1 were bred with males homozygous for floxed ET-1. Animals homozygous for floxed ET-1 and heterozygous for AQP2-Cre (CD ET-1 KO) were used in all studies. Sex-matched littermates that were homozygous for the floxed ET-1 gene, but without Cre, were used as controls in all studies.
Genotyping. Tail DNA was prepared by standard methods and PCR 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. Genotyping the ET-1 gene involved three primer sets. The first set used primers ET-1CF (5'-GCT GCC CAA AGA TTC TGA ATT C-3') and ET-1BR (5'-GAT GAT GTC CAG GTG GCA GAA G-3'), which amplify 800 bp of the endogenous ET-1 allele. The second set used primers ET-1AF (5'-CCC AAA GAT TCT GAA TTG ATA ACT TCG-3') and ET-1BR (5'-GAT GAT GTC CAG GTG GCA GAA G-3'), which amplify the same 800-bp region; however, the forward primer overlaps loxP and hence only recognizes the floxed ET-1 allele. The third set used primers ET-1AF and ET-1DR (5'-AAC CTC CCA GTC CAT ACG GTA C-3'), which amplify the region in the targeted allele spanning the loxP sites. The PCR product of the nonrecombined allele is 2 kb, whereas the recombined allele yields a 300-bp product. PCR products were visualized after electrophoresis through 1.5% agarose.
Metabolic balance studies. All mice were studied at 3–5 mo of age. All mice were acclimated for 1 wk to Nalgene metabolic cages. Mice were fed 9 ml of a gel diet that contained all nutrients and water. The gel food was made by dissolving 65.7 g of PMI rodent powder diet (LD101, Lab Diet, Richmond, IN) and 7 g of gelatin in 110 ml of water. NaCl was added to give a final sodium concentration of 0.3%. The gel was solidified in plastic scintillation vials and served as the sole source of food and water. 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. Mice were then killed by guillotine and blood was collected for plasma Na, osmolality, and AVP determination. Kidneys were removed for protein and mRNA determination.
For chronic water loading, mice were acclimatized to metabolic cages and a normal water gel diet for 1 wk, and balance studies as described above were performed for 3 more days, then switched to a high-water diet for up to 7 days. The high-water diet consisted of 19 ml of gel diet containing 14.1 ml of 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. Kidneys were removed for protein and mRNA determination.
1-Desamino-8-D-arginine vasopressin studies. Mice were acclimatized to metabolic cages, and 3 days of baseline measurements were taken as described above. Subsequently, mice were given 1-desamino-8-D-arginine vasopressin (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 gel diet). For combined DDAVP and water loading, mice were studied as above until the second day of DDAVP and then switched to the high water diet (containing 19 ml of gel 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, blood was obtained for electrolyte determination, and kidneys were removed for protein and mRNA measurements.
Acute water loading. Mice were acclimatized to metabolic cages and a gel normal water diet for 1 wk. They were then placed into small metabolic cages that contained no food or water and given 2 ml of water ip. Subsequently, urine was collected under oil on an hourly basis for the next 6 h. Urine was analyzed for volume and osmolality. Urine was then pooled for analysis of ET-1 excretion. In some studies, mice were killed at different time points after acute water loading, and the kidneys were removed for determination of IMCD ET-1 mRNA levels.
cAMP studies. IMCDs were acutely isolated in a manner similar to that previously described (21). Briefly, inner medullas were minced in Kreb's buffer with 1 mg/ml collagenase (Invitrogen, Carlsbad, CA) and 0.1 mg/ml DNase (Sigma) and incubated at 37°C for 30 min. IMCD fragments were washed and all subsequent incubations were done in Kreb's buffer. IMCDs were incubated in 1 mM isobutylmethylxanthine (Sigma) for 15 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, Minneapolis, MN) using a SpectraMax Plus Microplate Spectrophotometer (Molecular Devices, Sunnyvale, CA). Total cell protein was measured by the Bradford assay (Bio-Rad, Hercules, CA).
mRNA expression. Total RNA was prepared from kidneys using acid phenol. Twenty micrograms of RNA were electrophoresed on a 0.9% formaldehyde gel, transferred to a nylon membrane, and prehybridized for 3 h at 60°C in 50% formamide, 5x standard saline citrate, 5x Denhardt's solution, 1% SDS, and 100 μg/ml salmon sperm DNA. Fresh solution was added for hybridization along with radioactively labeled probe. For probes, cDNA was made from mouse IMCD cell total RNA using oligo dT mRNA primer and SuperScript II reverse transcriptase (Invitrogen). The cDNA was then used as a template for PCR amplification using specific primers as follows: AQP2 (GenBank accession no. NM-009699), forward primer 5-GTG GCT GCC CAG CTG CTG GG-3 and reverse primer 5-AGC TCC ACC GAC TGC CGC CG-3, which yields a product size of 509 bp; and V2 (GenBank accession no. NM-019404), forward primer 5-CAG TCA TTT GTG CCT AGC TGA CCT GG-3 and reverse primer 5-ATC ACT AGT GTC ATC CTC ACG G-3, which yields a product size of 596 bp. Products were purified, sequenced, and cloned into a pGEM-T cloning vector (Promega). The inserts were sequenced again to ensure cloning fidelity and to confirm orientation. Probes were digested with an appropriate restriction enzyme to give the antisense strand, and riboprobes were made by [32P]UTP incorporation with either T7 or SP6 RNA polymerase (Invitrogen). The radioactive probes were purified over a G-50 column, and specific activity was calculated. The probe was added to hybridization solution at 10 ng/ml with a specific activity 109 dpm/g and incubated overnight at 60°C. Blots were washed in decreasing concentrations of standard saline citrate and increasing temperature until background was removed. Labeled blots were subjected to autoradiography and densitometry. V2 RNA is 1.8 kb and AQP2 mRNA is 1.4 kb in size. All blots were stripped and reprobed for U1 small nuclear ribonucleoprotein (SNRP) mRNA (mRNA size is 0.75 kb) using a probe provided by Dr. A. Thorburn at the University of Utah (37).
For determination of IMCD ET-1 mRNA, IMCD fragments were obtained as described above. IMCDs were placed in GITC solution for acid phenol RNA isolation and reverse transcribed using oligo (dT)12–18 and Superscript II (Invitrogen). ET-1 cDNA was amplified with ET-1F 5'-GCT CCA GAA ACA GCT GTC TTG G-3' and ET-1R 5'-TTC TTT CCC AAC TTG GAA CAG GG-3', which yielded a 414-bp product. GAPDH was amplified with GAPDH-F 5'-CCT TCA TTG ACC TCA ACT ACA TGG-3' and GAPDH-R 5'-GCA GTG ATG GCA TGG ACT GTG GT-3', which yielded a 442-bp product. All PCR reactions were carried out with and without reverse transcription to check for genomic DNA contamination. PCR products were run on a 1.5% agarose gel, and products were quantified by densitometry.
Protein expression. Kidneys were minced and homogenized in 250 mM sucrose, 10 mM triethanolamine, and a 1:1,000 dilution of protease inhibitor cocktail set III (Calbiochem, La Jolla, CA). The sample was spun at 4,000 g for 15 min to remove nuclei, mitochondria, and larger cellular fragments. The supernatant containing the crude membrane fraction was centrifuged at 17,000 g for 30 min. Protein concentration was determined using the Bradford assay. Samples were solubilized at various concentrations in Laemmli buffer containing 0.5% lithium dodecyl sulfate and run on a denaturing NUPAGE 4–12% Bis-Tris minigel using the MOPS buffer system (Invitrogen). Proteins were transferred to PVDF plus nylon membranes by electroelution. The blots were blocked with 5% nonfat dry milk + TBST (10 mM Tris base, 150 mM NaCl, 0.1% Tween 20, pH 7.5) and then washed in TBST. Blots were incubated overnight at 4°C with the appropriate primary antibody, washed, and incubated with a horseradish peroxidase-conjugated (HRP) secondary antibody for 1 h. After final washes, antibody binding was visualized using the enhanced chemiluminescence system (Amersham International). Blots were stripped using 200 mM glycine, pH 2.8, washed with TBST, and reblocked before further probing. Antibodies used for total AQP2 were 1:500 goat anti-human AQP2 (Santa Cruz Biotechnology, Santa Cruz, CA) and 1:5,000 HRP-rabbit anti-goat IgG (Santa Cruz Biotechnology). Antibodies for phosphorylated AQP2 (P-AQP2) detection were 1:2,000 rabbit anti-rat P-AQP2 [AN-244-pp-AP (generously provided by S. Nielsen, University of Aarhus, Denmark, which recognizes Ser-256 phosphorylated AQP2) (8)] and 1:2,000 HRP-donkey anti-rabbit IgG (Amersham International). Antibodies against -actin were 1:10,000 rabbit anti-human -actin (Abcam, Cambridge, MA) and 1:2,000 HRP-donkey anti-rabbit IgG. All blots were reprobed for -actin.
Electrolyte and hormone analysis. Plasma from all animals was analyzed for Na concentration (EasyVet analyzer, Medica, Bedford, MA) and osmolality (Osmett II, Precision Systems, Natick, MA). Plasma from guillotined mice was also analyzed for AVP. For AVP determination, heparinized samples (100 μl) were mixed with 100 μl of water and 2 ml of ice-cold acetone, centrifuged for 20 min at 3,500 rpm, the supernatant was mixed with 2 ml of petroleum ether, and the bottom layer was lyophilized. AVP was assayed using a radioimmunoassay kit (Peninsula Laboratories, San Carlos, CA).
Urine was measured for osmolality and ET-1. Urine was applied to a 200-mg Bond Elut C8 column (Varian, Palo Alto, CA), equilibrated, eluted as previously described (34), and ET-1 was determined by ELISA (QuantiGlo, R&D Systems) using a Dynex Technologies MLX luminometer (Chantilly, VA).
Statistics and ethics. Comparisons between floxed ET-1 and CD ET-1 KO mice were analyzed by the unpaired Student's t-test. Comparisons of urine volume after acute water loading, AVP-stimulated cAMP accumulation, protein levels, mRNA levels, and daily weight change between floxed and CD ET-1 KO mice were made using one-way analysis of variance with the Bonferroni correction. P < 0.05 was taken as significant. Data are expressed as means ± SE. All animal experiments were ethically approved by the University of Utah Institutional Animal Care and Use Committee.
RESULTS
Characterization of CD ET-1 KO mice. CD ET-1 KO mice developed normally until at least 1 yr of age and had no gross morphological abnormalities. As previously described (1), CD-specific knockout of ET-1 was confirmed by in situ hybridization of kidney for ET-1 mRNA, genomic PCR of microdissected CD for evidence of ET-1 gene recombination, genomic PCR for ET-1 gene recombination in an organ panel of 15 different organs, determination of renal cell Cre activity in mice heterozygous for AQP2-Cre and the ROSA26-YFP reporter, and measurement of urinary ET-1. Thus CD ET-1 KO mice have CD-specific inactivation of the ET-1 gene.
Renal function during a normal water 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 gel 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 urine volume, urine or plasma osmolality, or plasma Na concentration (Table 1). Mice have high insensible water loss, so urine volume is much less than water intake. As previously reported (1), creatinine clearance was similar between floxed ET-1 and CD ET-1 KO mice (data not shown). Notably, plasma AVP concentration was substantially less in CD ET-1 KO mice, despite all other aspects of water metabolism being similar (Table 1). 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 lower plasma AVP levels.
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Renal function during high water intake. CD-derived ET-1 may be important in mediating the diuretic response to increased water intake. To assess this, CD ET-1 KO mice were chronically water loaded (19 ml of gel diet containing 14.1 ml of water). Mice ate all the gel (they ate more gel than provided with the control diet due to the lower concentration of nutrients in the water-loading diet) and had closely matched intakes of water (14.0 ± 0.2 ml in controls and 14.1 ± 0.2 ml in CD ET-1 KO). As shown in Fig. 1, there was no difference in urine volume or urine osmolality between CD ET-1 KO and control mice for up to 7 days of increased water intake. Similarly, after the 7 days of water loading, there was no difference in plasma Na concentration or plasma osmolality between the two groups, whereas plasma AVP levels were suppressed in both groups to essentially the lower limits of detectability and not significantly different from zero (Table 2). Thus CD ET-1 KO mice have no apparent defect in their ability to handle a chronic water load.
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Renal function during DDAVP administration. The reduced plasma AVP levels during a normal water diet in CD ET-1 KO mice suggested that these mice have enhanced responsiveness to the hydrosmotic effects of AVP. To test this, mice were given DDAVP via an osmotic minipump for up to 7 days, while being on a normal water intake (9 ml of the normal water intake gel diet). This procedure was designed to fix plasma AVP at maximal water-retaining levels. Chronic DDAVP administration significantly increased urine osmolality in CD ET-1 KO mice but not in control animals (Fig. 2). The failure to see an increase in urine osmolality in controls was assumed to relate to the relatively hydropenic state under which the mice must normally exist. Notably, urine osmolality did not remain elevated in CD ET-1 KO mice beyond day 3 of DDAVP administration, indicating that compensatory mechanisms came into play. No significant decrease in urine volume could be detected in either group of mice; however, such a decrease would be difficult to detect due to the intrinsic high variability in urine volume within groups. However, CD ET-1 KO mice did have more weight gain, at least on day 2 of DDAVP administration, compared with controls, indicating that they must have retained more fluid than controls (Fig. 3). CD ET-1 KO mice tended to have lower plasma Na concentration (130 ± 3 meq/l in CD ET-1 KO vs. 134.2 ± 3 meq/l in controls) and plasma osmolality (301 ± 4 in CD ET-1 KO vs. 305 ± 5 in controls), but this did not achieve statistical significance. Thus these data suggested that CD ET-1 KO mice do, in fact, have an enhanced antidiuretic response to AVP.
The above studies raised the question as to whether CD ET-1 KO mice have impaired AVP "escape," i.e., have less decrease in urine osmolality and less increase in urine volume in response to combined DDAVP administration and water loading. As shown in Figs. 3 and 4, there was no difference in urine volume, urine osmolality, or weight gain between control and CD ET-1 KO mice on any day during the 5 days of water loading during DDAVP administration. There was also no significant difference in plasma Na concentration or plasma osmolality (data not shown). Thus CD ET-1 KO does not impair the ability to escape from the hydrosmotic effects of DDAVP.
Effect of CD ET-1 KO on V2 receptor and AQP2 expression. Given that CD ET-1 KO did not change urine volume or urine osmolality under basal or high water intake conditions, it seemed unlikely that either V2 or AQP2 expression would be altered in these mice. However, to confirm this, membrane-associated AQP2 and phosphorylated AQP2 protein, AQP2 mRNA, and V2 mRNA (there are no reliable antibodies to V2) levels were determined. As expected, total and phosphorylated AQP2 protein levels were not altered in CD ET-1 KO mice, compared with controls, on a normal water diet or after 7 days of water loading (Fig. 5). V2 mRNA levels were also not affected; however, AQP2 mRNA was slightly, albeit significantly, increased in CD ET-1 KO mice, compared with controls, on a normal water diet (Fig. 6). Similar studies were performed in mice given DDAVP. Of note, DDAVP administration to mice on a normal water diet (wherein CD ET-1 KO mice had increased urine osmolality and weight gain compared with controls) resulted in increases in phosphorylated AQP2, V2 mRNA, and AQP2 mRNA levels (compared with controls) (Figs. 5 and 6). Concurrent water loading and DDAVP administration for 5 days resulted in no difference in any of these parameters between CD ET-1 KO and control mice (Figs. 5 and 6). Thus these data again suggest enhanced AVP responsiveness in CD ET-1 KO mice on a normal water diet.
Effect of acute water loading. Although CD ET-1 KO mice exhibited increased AVP responsiveness, no data thus far indicated that this was of particular physiological relevance. Based on previous studies discussed earlier, it seemed most likely that CD-derived ET-1 would likely help mediate the diuretic response to water loading. A significant problem with our water-loading studies was that they 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. Indeed, as illustrated in Fig. 7, CD ET-1 KO mice had an impaired response to acute water loading (2 ml ip). In particular, ET-1 KO mice had reduced water excretion, compared with controls, up to 3 h after an acute water load. These data suggest, therefore, that CD-derived ET-1 plays a role in mediating the diuretic response to water loading, at least within the first several hours of increased water intake.
If CD ET-1 were involved in mediating, at least in part, the diuretic response to an acute water load, then it would be reasonable to expect an increase in CD ET-1 production during this acute phase. To first assess this, urinary ET-1 excretion was determined after an acute water load. Because renal ET-1 excretion is relatively low in mice, it was possible to only accurately assess urinary ET-1 excretion for the entire 6-h period after water loading (i.e., not on an hourly basis). ET-1 KO mice excreted markedly less ET-1 than did controls over the 6-h period (2.46 ± 0.45 pg ET-1 for CD ET-1 KO and 4.85 ± 0.93 pg ET-1 for controls). To more directly assess CD ET-1 production, IMCDs were acutely isolated from control kidneys after acute water loading [CD ET-1 KO IMCD do not have ET-1 mRNA (1)]. As shown in Fig. 8, there was a qualitative increase in IMCD ET-1 mRNA levels that were first evident 2 h after water loading and persisted up to at least 6 h. Thus these data provide further evidence that CD-derived ET-1 plays a role in the diuretic response to an acute water load.
Agonist-stimulated cAMP accumulation. To more directly assess increased CD sensitivity to AVP in CD ET-1 KO mice, IMCDs were acutely isolated and stimulated with AVP. Note that outer MCDs or cortical CDs were not studied due to inability to acutely isolate sufficient quantities of these nephron segments. IMCD from CD ET-1 KO kidneys had significantly greater AVP-stimulated cAMP accumulation, and this occurred at AVP concentrations as low as 100 pM (Fig. 9). To determine whether this augmented AVP response was due, at least in part, to postreceptor mechanisms, IMCDs were stimulated with forskolin. Similar to AVP, forskolin increased cAMP levels much more in IMCD from CD ET-1 KO mice compared with controls (Fig. 9). Thus at least part of the enhanced AVP responsiveness observed in vivo can be related to increased AVP-induced CD cAMP accumulation via postreceptor mechanisms.
DISCUSSION
The present study demonstrates that CD-derived ET-1 regulates renal water excretion. CD-specific knockout of ET-1 reduces plasma AVP levels yet does not alter renal water excretion under baseline conditions or in response to a chronic water load. Thus, while the relationship between plasma AVP levels and urine osmolality was reset, other mechanisms were able to fully compensate. However, when plasma AVP levels are not permitted to reset by exogenous AVP administration, CD ET-1 KO mice exhibit enhanced urine osmolality. The physiological relevance of this ET-1-mediated downregulation of AVP responsiveness was apparent during acute water loading, wherein CD ET-1 KO mice had impaired ability to mount a diuresis. Increased AVP responsiveness was further confirmed by demonstrating enhanced AVP-induced cAMP accumulation in acutely isolated CD ET-1 KO IMCD. Taken together, these studies provide the first demonstration of altered AVP responsiveness due to deficiency of a peptide not directly involved in water transport.
How absence of CD-derived ET-1 leads to enhanced AVP responsiveness and a reduced acute diuretic response is not yet fully understood. Previous in vitro studies suggested that CD-derived ET-1 could act in an autocrine manner by activating CD ETRB with resultant decreased AVP-stimulated cAMP accumulation (10, 21, 39). Our finding that CD ET-1 KO mice also have increased forskolin-stimulated cAMP accumulation in acutely isolated IMCD is in agreement with previous in vitro studies (21) and suggests that enhanced AVP responsiveness in CD ET-1 KO IMCD is due, at least partially, to postreceptor mechanisms. Which mechanisms are involved in such altered signaling remains to be determined, although potential candidates have been identified. For example, ET-1 may increase CD nitric oxide (NO) production (45), whereas NO reduces AVP-stimulated water permeability in isolated cortical CD (12). The increased AVP responsiveness is associated with augmented AQP2 phosphorylation and elevated mRNA levels for the V2 receptor and AQP2 (as seen in kidneys from CD ET-1 KO mice given DDAVP and a normal water diet). These changes may well be related to increased AVP-induced cAMP accumulation; however, this was not directly assessed. It is possible that CD ET-1 KO leads to increased AVP binding, through altered V2 receptor expression or affinity, which could partially account for increased AVP responsiveness. This could be tested using radiolabeled AVP analog binding to acutely isolated IMCD; such studies are planned as part of follow-up investigations on the mechanism of CD ET-1 KO-mediated alterations in AVP responsiveness.
CD-derived ET-1 could also modulate water reabsorption through paracrine effects. Adjacent medullary interstitial cells express ET receptors (9) but do not synthesize ET-1 (43). ET-1 elicits cultured medullary interstitial cell NO production (47), which could conceivably act on neighboring CD to inhibit water reabsorption. In addition, medullary CD increases in ET-1 release might lead to augmented medullary blood flow (4, 11), thereby washing out the medullary gradient and facilitating water excretion.
The factors responsible for augmenting CD ET-1 production within a few hours of water loading are unknown. Hypertonicity has been found to reduce cultured CD ET-1 production (20, 33, 38), although this has not been consistently noted (26, 44). Volume depletion, which increases medullary tonicity, decreases renal medullary ET-1 content, whereas volume expansion increases medullary ET-1 levels (20). Another interesting possibility is that increased tubule fluid flow rate may regulate CD ET-1 production. Cai et al. (5) reported that shear stress increases all three NO synthase isoforms in cultured IMCD; the possibility is raised that ET-1 could also be increased and may, at least in part, lead to increased NO production. Given that the IMCD is the major nephron source of ET-1 and that the IMCD is a major site of AVP-stimulated water reabsorption, clarification of this regulatory system is of substantial importance.
In summary, the present studies demonstrate that CD-derived ET-1 is involved in the acute diuretic response to water loading and chronically modulates CD AVP responsiveness. In contrast, CD-derived ET-1 is not necessary for a maximal diuretic response to water loading nor does it play a significant role in mediating AVP escape. Further studies are needed on the mechanisms by which CD-derived ET-1 is regulated and the mechanisms by which it regulates renal water excretion.
GRANTS
This research was funded in part by National Institutes of Health Grants DK-59047 and DK-58953 to D. E. Kohan.
DISCLOSURES
There is no conflict of interest for these studies.
FOOTNOTES
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.
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Department of Physiology, Kosin University College of Medicine, Kosin, South Korea
Howard Hughes Medical Institute, University of Texas Southwestern, Dallas, Texas
Division of Endocrinology and Metabolism, Georgetown University, Washington, District of Columbia
Salt Lake Veterans Affairs Medical Center, Salt Lake City, Utah
ABSTRACT
In vitro studies suggest that endothelin-1 (ET-1) inhibits vasopressin (AVP)-stimulated water permeability in the collecting duct (CD). To evaluate the role of CD-derived ET-1 in regulating renal water metabolism, the ET-1 gene was selectively disrupted in the CD (CD ET-1 KO). During normal water intake, urinary osmolality (Uosm), plasma Na concentration, urine volume, and renal aquaporin-2 (AQP2) levels were unchanged, but plasma AVP concentration was reduced in CD ET-1 KO animals. CD ET-1 KO mice had impaired ability to excrete an acute, but not a chronic, water load, and this was associated with increased CD ET-1 mRNA in control, but not CD ET-1 KO, mice. In response to continuous infusion of 1-desamino-8-D-arginine vasopressin, CD ET-1 KO mice had greater increases in Uosm, V2 and AQP2 mRNA, and phosphorylation of AQP2. CD suspensions from CD ET-1 KO mice had enhanced AVP- and forskolin-stimulated cAMP accumulation. These data indicate that CD ET-1 KO increases renal sensitivity to the urinary concentrating effects of AVP and suggest that ET-1 functions as a physiological autocrine regulator of AVP action in the CD.
aquaporin-2; adenosine 3',5'-cyclic monophosphate; inner medullary collecting duct
IN VITRO STUDIES SUGGEST THAT collecting duct (CD)-derived endothelin-1 (ET-1) may be an autocrine inhibitor of vasopressin (AVP)-stimulated water reabsorption in the kidney. Within the kidney, the CD is the major renal site of ET-1 synthesis (6, 18, 31, 41, 42) and possibly binding (7, 35, 36). In vitro, exogenous ET-1, through activation of the ETB receptor (ETRB), reduces AVP-stimulated water permeability in cortical CD (35, 40) and inhibits AVP-stimulated cAMP accumulation (10, 21, 39) and osmotic water permeability (27, 29) in inner medullary CD (IMCD). Blockade of endogenous ET-1 action increases AVP-stimulated cAMP levels in cultured IMCD cells (19), providing the most direct evidence for a potential autocrine role of the peptide.
There is less convincing, and no direct, evidence demonstrating an autocrine role for ET-1 in regulating CD water transport in vivo. Part of the problem stems from the inability to discriminate between direct ET-1 effects on the nephron as opposed to the vasculature. Administration of ET agonists or antagonists usually alters renal plasma flow and glomerular filtration rate, effects that can impact urinary water excretion. Studies have shown that (17) systemically administered ET, at doses with minimal detectable effects on renal hemodynamics, increases water excretion (14, 16, 32). Additionally, chronic water loading in rats and humans is associated with elevated urinary ET-1 excretion (although the source of urinary ET-1 could not be fully ascertained) (20, 24, 46), whereas water deprivation decreases urinary ET-1 excretion (20, 25). Thus circumstantial evidence suggests that ET-1 may inhibit renal water reabsorption. However, the physiological role that the peptide plays in regulating renal water transport, particularly through autocrine regulation of CD function, remains unknown.
Alternative methods for examining the physiological role of CD-derived ET-1 in regulating renal water excretion have not yet been employed. Traditional gene knockout methodologies have proven problematic in that every mouse homozygous for knockout of a component of the ET system [ET-1, ET-2, ET-3, ET receptor A (ETRA), ETRB] has a lethal phenotype (2, 15, 22, 23, 30). Renal function has been assessed on a limited basis in spotted lethal rats, which carry a natural mutation in the ETRB gene resulting in toxic megacolon (13). Spotted lethal rats that have been "rescued" with a transgene expressing ETRB under control of the dopamine -hydroxylase promoter exhibit hypertension; however, renal water excretion has not been reported in this model. Furthermore, these animals lack ETRB expression in the kidney [almost every cell in the kidney expresses ETRB (17)], while circulating ET-1 levels are likely to be markedly elevated (3). Consequently, this model was not employed. We recently reported the development of mice with CD-specific knockout of ET-1. These mice express Cre recombinase under control of the aquaporin-2 (AQP2) promoter and are homozygous for loxP-flanked exon 2 of the ET-1 gene. We now report that these mice have altered renal water handling, thereby providing direct evidence for a physiological role of CD-derived ET-1 in regulating renal water excretion.
METHODS
Transgenic mice lines. Mice with CD-specific disruption of the ET-1 gene were generated in a manner similar to that previously described (1). Briefly, mice containing the loxP-flanked (floxed) ET-1 gene (obtained from Dr. M. Yanagisawa at the Howard Hughes Institute at University of Texas Southwestern Medical Center) were mated with AQP2-Cre mice. The floxed mice contain exon 2 of the ET-1 gene flanked by loxP sites [exon 2 is critical to ET-1 gene functional expression (23)]. 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 (28). 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 ET-1 mice; female offspring heterozygous for both AQP2-Cre and floxed ET-1 were bred with males homozygous for floxed ET-1. Animals homozygous for floxed ET-1 and heterozygous for AQP2-Cre (CD ET-1 KO) were used in all studies. Sex-matched littermates that were homozygous for the floxed ET-1 gene, but without Cre, were used as controls in all studies.
Genotyping. Tail DNA was prepared by standard methods and PCR 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. Genotyping the ET-1 gene involved three primer sets. The first set used primers ET-1CF (5'-GCT GCC CAA AGA TTC TGA ATT C-3') and ET-1BR (5'-GAT GAT GTC CAG GTG GCA GAA G-3'), which amplify 800 bp of the endogenous ET-1 allele. The second set used primers ET-1AF (5'-CCC AAA GAT TCT GAA TTG ATA ACT TCG-3') and ET-1BR (5'-GAT GAT GTC CAG GTG GCA GAA G-3'), which amplify the same 800-bp region; however, the forward primer overlaps loxP and hence only recognizes the floxed ET-1 allele. The third set used primers ET-1AF and ET-1DR (5'-AAC CTC CCA GTC CAT ACG GTA C-3'), which amplify the region in the targeted allele spanning the loxP sites. The PCR product of the nonrecombined allele is 2 kb, whereas the recombined allele yields a 300-bp product. PCR products were visualized after electrophoresis through 1.5% agarose.
Metabolic balance studies. All mice were studied at 3–5 mo of age. All mice were acclimated for 1 wk to Nalgene metabolic cages. Mice were fed 9 ml of a gel diet that contained all nutrients and water. The gel food was made by dissolving 65.7 g of PMI rodent powder diet (LD101, Lab Diet, Richmond, IN) and 7 g of gelatin in 110 ml of water. NaCl was added to give a final sodium concentration of 0.3%. The gel was solidified in plastic scintillation vials and served as the sole source of food and water. 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. Mice were then killed by guillotine and blood was collected for plasma Na, osmolality, and AVP determination. Kidneys were removed for protein and mRNA determination.
For chronic water loading, mice were acclimatized to metabolic cages and a normal water gel diet for 1 wk, and balance studies as described above were performed for 3 more days, then switched to a high-water diet for up to 7 days. The high-water diet consisted of 19 ml of gel diet containing 14.1 ml of 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. Kidneys were removed for protein and mRNA determination.
1-Desamino-8-D-arginine vasopressin studies. Mice were acclimatized to metabolic cages, and 3 days of baseline measurements were taken as described above. Subsequently, mice were given 1-desamino-8-D-arginine vasopressin (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 gel diet). For combined DDAVP and water loading, mice were studied as above until the second day of DDAVP and then switched to the high water diet (containing 19 ml of gel 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, blood was obtained for electrolyte determination, and kidneys were removed for protein and mRNA measurements.
Acute water loading. Mice were acclimatized to metabolic cages and a gel normal water diet for 1 wk. They were then placed into small metabolic cages that contained no food or water and given 2 ml of water ip. Subsequently, urine was collected under oil on an hourly basis for the next 6 h. Urine was analyzed for volume and osmolality. Urine was then pooled for analysis of ET-1 excretion. In some studies, mice were killed at different time points after acute water loading, and the kidneys were removed for determination of IMCD ET-1 mRNA levels.
cAMP studies. IMCDs were acutely isolated in a manner similar to that previously described (21). Briefly, inner medullas were minced in Kreb's buffer with 1 mg/ml collagenase (Invitrogen, Carlsbad, CA) and 0.1 mg/ml DNase (Sigma) and incubated at 37°C for 30 min. IMCD fragments were washed and all subsequent incubations were done in Kreb's buffer. IMCDs were incubated in 1 mM isobutylmethylxanthine (Sigma) for 15 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, Minneapolis, MN) using a SpectraMax Plus Microplate Spectrophotometer (Molecular Devices, Sunnyvale, CA). Total cell protein was measured by the Bradford assay (Bio-Rad, Hercules, CA).
mRNA expression. Total RNA was prepared from kidneys using acid phenol. Twenty micrograms of RNA were electrophoresed on a 0.9% formaldehyde gel, transferred to a nylon membrane, and prehybridized for 3 h at 60°C in 50% formamide, 5x standard saline citrate, 5x Denhardt's solution, 1% SDS, and 100 μg/ml salmon sperm DNA. Fresh solution was added for hybridization along with radioactively labeled probe. For probes, cDNA was made from mouse IMCD cell total RNA using oligo dT mRNA primer and SuperScript II reverse transcriptase (Invitrogen). The cDNA was then used as a template for PCR amplification using specific primers as follows: AQP2 (GenBank accession no. NM-009699), forward primer 5-GTG GCT GCC CAG CTG CTG GG-3 and reverse primer 5-AGC TCC ACC GAC TGC CGC CG-3, which yields a product size of 509 bp; and V2 (GenBank accession no. NM-019404), forward primer 5-CAG TCA TTT GTG CCT AGC TGA CCT GG-3 and reverse primer 5-ATC ACT AGT GTC ATC CTC ACG G-3, which yields a product size of 596 bp. Products were purified, sequenced, and cloned into a pGEM-T cloning vector (Promega). The inserts were sequenced again to ensure cloning fidelity and to confirm orientation. Probes were digested with an appropriate restriction enzyme to give the antisense strand, and riboprobes were made by [32P]UTP incorporation with either T7 or SP6 RNA polymerase (Invitrogen). The radioactive probes were purified over a G-50 column, and specific activity was calculated. The probe was added to hybridization solution at 10 ng/ml with a specific activity 109 dpm/g and incubated overnight at 60°C. Blots were washed in decreasing concentrations of standard saline citrate and increasing temperature until background was removed. Labeled blots were subjected to autoradiography and densitometry. V2 RNA is 1.8 kb and AQP2 mRNA is 1.4 kb in size. All blots were stripped and reprobed for U1 small nuclear ribonucleoprotein (SNRP) mRNA (mRNA size is 0.75 kb) using a probe provided by Dr. A. Thorburn at the University of Utah (37).
For determination of IMCD ET-1 mRNA, IMCD fragments were obtained as described above. IMCDs were placed in GITC solution for acid phenol RNA isolation and reverse transcribed using oligo (dT)12–18 and Superscript II (Invitrogen). ET-1 cDNA was amplified with ET-1F 5'-GCT CCA GAA ACA GCT GTC TTG G-3' and ET-1R 5'-TTC TTT CCC AAC TTG GAA CAG GG-3', which yielded a 414-bp product. GAPDH was amplified with GAPDH-F 5'-CCT TCA TTG ACC TCA ACT ACA TGG-3' and GAPDH-R 5'-GCA GTG ATG GCA TGG ACT GTG GT-3', which yielded a 442-bp product. All PCR reactions were carried out with and without reverse transcription to check for genomic DNA contamination. PCR products were run on a 1.5% agarose gel, and products were quantified by densitometry.
Protein expression. Kidneys were minced and homogenized in 250 mM sucrose, 10 mM triethanolamine, and a 1:1,000 dilution of protease inhibitor cocktail set III (Calbiochem, La Jolla, CA). The sample was spun at 4,000 g for 15 min to remove nuclei, mitochondria, and larger cellular fragments. The supernatant containing the crude membrane fraction was centrifuged at 17,000 g for 30 min. Protein concentration was determined using the Bradford assay. Samples were solubilized at various concentrations in Laemmli buffer containing 0.5% lithium dodecyl sulfate and run on a denaturing NUPAGE 4–12% Bis-Tris minigel using the MOPS buffer system (Invitrogen). Proteins were transferred to PVDF plus nylon membranes by electroelution. The blots were blocked with 5% nonfat dry milk + TBST (10 mM Tris base, 150 mM NaCl, 0.1% Tween 20, pH 7.5) and then washed in TBST. Blots were incubated overnight at 4°C with the appropriate primary antibody, washed, and incubated with a horseradish peroxidase-conjugated (HRP) secondary antibody for 1 h. After final washes, antibody binding was visualized using the enhanced chemiluminescence system (Amersham International). Blots were stripped using 200 mM glycine, pH 2.8, washed with TBST, and reblocked before further probing. Antibodies used for total AQP2 were 1:500 goat anti-human AQP2 (Santa Cruz Biotechnology, Santa Cruz, CA) and 1:5,000 HRP-rabbit anti-goat IgG (Santa Cruz Biotechnology). Antibodies for phosphorylated AQP2 (P-AQP2) detection were 1:2,000 rabbit anti-rat P-AQP2 [AN-244-pp-AP (generously provided by S. Nielsen, University of Aarhus, Denmark, which recognizes Ser-256 phosphorylated AQP2) (8)] and 1:2,000 HRP-donkey anti-rabbit IgG (Amersham International). Antibodies against -actin were 1:10,000 rabbit anti-human -actin (Abcam, Cambridge, MA) and 1:2,000 HRP-donkey anti-rabbit IgG. All blots were reprobed for -actin.
Electrolyte and hormone analysis. Plasma from all animals was analyzed for Na concentration (EasyVet analyzer, Medica, Bedford, MA) and osmolality (Osmett II, Precision Systems, Natick, MA). Plasma from guillotined mice was also analyzed for AVP. For AVP determination, heparinized samples (100 μl) were mixed with 100 μl of water and 2 ml of ice-cold acetone, centrifuged for 20 min at 3,500 rpm, the supernatant was mixed with 2 ml of petroleum ether, and the bottom layer was lyophilized. AVP was assayed using a radioimmunoassay kit (Peninsula Laboratories, San Carlos, CA).
Urine was measured for osmolality and ET-1. Urine was applied to a 200-mg Bond Elut C8 column (Varian, Palo Alto, CA), equilibrated, eluted as previously described (34), and ET-1 was determined by ELISA (QuantiGlo, R&D Systems) using a Dynex Technologies MLX luminometer (Chantilly, VA).
Statistics and ethics. Comparisons between floxed ET-1 and CD ET-1 KO mice were analyzed by the unpaired Student's t-test. Comparisons of urine volume after acute water loading, AVP-stimulated cAMP accumulation, protein levels, mRNA levels, and daily weight change between floxed and CD ET-1 KO mice were made using one-way analysis of variance with the Bonferroni correction. P < 0.05 was taken as significant. Data are expressed as means ± SE. All animal experiments were ethically approved by the University of Utah Institutional Animal Care and Use Committee.
RESULTS
Characterization of CD ET-1 KO mice. CD ET-1 KO mice developed normally until at least 1 yr of age and had no gross morphological abnormalities. As previously described (1), CD-specific knockout of ET-1 was confirmed by in situ hybridization of kidney for ET-1 mRNA, genomic PCR of microdissected CD for evidence of ET-1 gene recombination, genomic PCR for ET-1 gene recombination in an organ panel of 15 different organs, determination of renal cell Cre activity in mice heterozygous for AQP2-Cre and the ROSA26-YFP reporter, and measurement of urinary ET-1. Thus CD ET-1 KO mice have CD-specific inactivation of the ET-1 gene.
Renal function during a normal water 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 gel 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 urine volume, urine or plasma osmolality, or plasma Na concentration (Table 1). Mice have high insensible water loss, so urine volume is much less than water intake. As previously reported (1), creatinine clearance was similar between floxed ET-1 and CD ET-1 KO mice (data not shown). Notably, plasma AVP concentration was substantially less in CD ET-1 KO mice, despite all other aspects of water metabolism being similar (Table 1). 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 lower plasma AVP levels.
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Renal function during high water intake. CD-derived ET-1 may be important in mediating the diuretic response to increased water intake. To assess this, CD ET-1 KO mice were chronically water loaded (19 ml of gel diet containing 14.1 ml of water). Mice ate all the gel (they ate more gel than provided with the control diet due to the lower concentration of nutrients in the water-loading diet) and had closely matched intakes of water (14.0 ± 0.2 ml in controls and 14.1 ± 0.2 ml in CD ET-1 KO). As shown in Fig. 1, there was no difference in urine volume or urine osmolality between CD ET-1 KO and control mice for up to 7 days of increased water intake. Similarly, after the 7 days of water loading, there was no difference in plasma Na concentration or plasma osmolality between the two groups, whereas plasma AVP levels were suppressed in both groups to essentially the lower limits of detectability and not significantly different from zero (Table 2). Thus CD ET-1 KO mice have no apparent defect in their ability to handle a chronic water load.
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Renal function during DDAVP administration. The reduced plasma AVP levels during a normal water diet in CD ET-1 KO mice suggested that these mice have enhanced responsiveness to the hydrosmotic effects of AVP. To test this, mice were given DDAVP via an osmotic minipump for up to 7 days, while being on a normal water intake (9 ml of the normal water intake gel diet). This procedure was designed to fix plasma AVP at maximal water-retaining levels. Chronic DDAVP administration significantly increased urine osmolality in CD ET-1 KO mice but not in control animals (Fig. 2). The failure to see an increase in urine osmolality in controls was assumed to relate to the relatively hydropenic state under which the mice must normally exist. Notably, urine osmolality did not remain elevated in CD ET-1 KO mice beyond day 3 of DDAVP administration, indicating that compensatory mechanisms came into play. No significant decrease in urine volume could be detected in either group of mice; however, such a decrease would be difficult to detect due to the intrinsic high variability in urine volume within groups. However, CD ET-1 KO mice did have more weight gain, at least on day 2 of DDAVP administration, compared with controls, indicating that they must have retained more fluid than controls (Fig. 3). CD ET-1 KO mice tended to have lower plasma Na concentration (130 ± 3 meq/l in CD ET-1 KO vs. 134.2 ± 3 meq/l in controls) and plasma osmolality (301 ± 4 in CD ET-1 KO vs. 305 ± 5 in controls), but this did not achieve statistical significance. Thus these data suggested that CD ET-1 KO mice do, in fact, have an enhanced antidiuretic response to AVP.
The above studies raised the question as to whether CD ET-1 KO mice have impaired AVP "escape," i.e., have less decrease in urine osmolality and less increase in urine volume in response to combined DDAVP administration and water loading. As shown in Figs. 3 and 4, there was no difference in urine volume, urine osmolality, or weight gain between control and CD ET-1 KO mice on any day during the 5 days of water loading during DDAVP administration. There was also no significant difference in plasma Na concentration or plasma osmolality (data not shown). Thus CD ET-1 KO does not impair the ability to escape from the hydrosmotic effects of DDAVP.
Effect of CD ET-1 KO on V2 receptor and AQP2 expression. Given that CD ET-1 KO did not change urine volume or urine osmolality under basal or high water intake conditions, it seemed unlikely that either V2 or AQP2 expression would be altered in these mice. However, to confirm this, membrane-associated AQP2 and phosphorylated AQP2 protein, AQP2 mRNA, and V2 mRNA (there are no reliable antibodies to V2) levels were determined. As expected, total and phosphorylated AQP2 protein levels were not altered in CD ET-1 KO mice, compared with controls, on a normal water diet or after 7 days of water loading (Fig. 5). V2 mRNA levels were also not affected; however, AQP2 mRNA was slightly, albeit significantly, increased in CD ET-1 KO mice, compared with controls, on a normal water diet (Fig. 6). Similar studies were performed in mice given DDAVP. Of note, DDAVP administration to mice on a normal water diet (wherein CD ET-1 KO mice had increased urine osmolality and weight gain compared with controls) resulted in increases in phosphorylated AQP2, V2 mRNA, and AQP2 mRNA levels (compared with controls) (Figs. 5 and 6). Concurrent water loading and DDAVP administration for 5 days resulted in no difference in any of these parameters between CD ET-1 KO and control mice (Figs. 5 and 6). Thus these data again suggest enhanced AVP responsiveness in CD ET-1 KO mice on a normal water diet.
Effect of acute water loading. Although CD ET-1 KO mice exhibited increased AVP responsiveness, no data thus far indicated that this was of particular physiological relevance. Based on previous studies discussed earlier, it seemed most likely that CD-derived ET-1 would likely help mediate the diuretic response to water loading. A significant problem with our water-loading studies was that they 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. Indeed, as illustrated in Fig. 7, CD ET-1 KO mice had an impaired response to acute water loading (2 ml ip). In particular, ET-1 KO mice had reduced water excretion, compared with controls, up to 3 h after an acute water load. These data suggest, therefore, that CD-derived ET-1 plays a role in mediating the diuretic response to water loading, at least within the first several hours of increased water intake.
If CD ET-1 were involved in mediating, at least in part, the diuretic response to an acute water load, then it would be reasonable to expect an increase in CD ET-1 production during this acute phase. To first assess this, urinary ET-1 excretion was determined after an acute water load. Because renal ET-1 excretion is relatively low in mice, it was possible to only accurately assess urinary ET-1 excretion for the entire 6-h period after water loading (i.e., not on an hourly basis). ET-1 KO mice excreted markedly less ET-1 than did controls over the 6-h period (2.46 ± 0.45 pg ET-1 for CD ET-1 KO and 4.85 ± 0.93 pg ET-1 for controls). To more directly assess CD ET-1 production, IMCDs were acutely isolated from control kidneys after acute water loading [CD ET-1 KO IMCD do not have ET-1 mRNA (1)]. As shown in Fig. 8, there was a qualitative increase in IMCD ET-1 mRNA levels that were first evident 2 h after water loading and persisted up to at least 6 h. Thus these data provide further evidence that CD-derived ET-1 plays a role in the diuretic response to an acute water load.
Agonist-stimulated cAMP accumulation. To more directly assess increased CD sensitivity to AVP in CD ET-1 KO mice, IMCDs were acutely isolated and stimulated with AVP. Note that outer MCDs or cortical CDs were not studied due to inability to acutely isolate sufficient quantities of these nephron segments. IMCD from CD ET-1 KO kidneys had significantly greater AVP-stimulated cAMP accumulation, and this occurred at AVP concentrations as low as 100 pM (Fig. 9). To determine whether this augmented AVP response was due, at least in part, to postreceptor mechanisms, IMCDs were stimulated with forskolin. Similar to AVP, forskolin increased cAMP levels much more in IMCD from CD ET-1 KO mice compared with controls (Fig. 9). Thus at least part of the enhanced AVP responsiveness observed in vivo can be related to increased AVP-induced CD cAMP accumulation via postreceptor mechanisms.
DISCUSSION
The present study demonstrates that CD-derived ET-1 regulates renal water excretion. CD-specific knockout of ET-1 reduces plasma AVP levels yet does not alter renal water excretion under baseline conditions or in response to a chronic water load. Thus, while the relationship between plasma AVP levels and urine osmolality was reset, other mechanisms were able to fully compensate. However, when plasma AVP levels are not permitted to reset by exogenous AVP administration, CD ET-1 KO mice exhibit enhanced urine osmolality. The physiological relevance of this ET-1-mediated downregulation of AVP responsiveness was apparent during acute water loading, wherein CD ET-1 KO mice had impaired ability to mount a diuresis. Increased AVP responsiveness was further confirmed by demonstrating enhanced AVP-induced cAMP accumulation in acutely isolated CD ET-1 KO IMCD. Taken together, these studies provide the first demonstration of altered AVP responsiveness due to deficiency of a peptide not directly involved in water transport.
How absence of CD-derived ET-1 leads to enhanced AVP responsiveness and a reduced acute diuretic response is not yet fully understood. Previous in vitro studies suggested that CD-derived ET-1 could act in an autocrine manner by activating CD ETRB with resultant decreased AVP-stimulated cAMP accumulation (10, 21, 39). Our finding that CD ET-1 KO mice also have increased forskolin-stimulated cAMP accumulation in acutely isolated IMCD is in agreement with previous in vitro studies (21) and suggests that enhanced AVP responsiveness in CD ET-1 KO IMCD is due, at least partially, to postreceptor mechanisms. Which mechanisms are involved in such altered signaling remains to be determined, although potential candidates have been identified. For example, ET-1 may increase CD nitric oxide (NO) production (45), whereas NO reduces AVP-stimulated water permeability in isolated cortical CD (12). The increased AVP responsiveness is associated with augmented AQP2 phosphorylation and elevated mRNA levels for the V2 receptor and AQP2 (as seen in kidneys from CD ET-1 KO mice given DDAVP and a normal water diet). These changes may well be related to increased AVP-induced cAMP accumulation; however, this was not directly assessed. It is possible that CD ET-1 KO leads to increased AVP binding, through altered V2 receptor expression or affinity, which could partially account for increased AVP responsiveness. This could be tested using radiolabeled AVP analog binding to acutely isolated IMCD; such studies are planned as part of follow-up investigations on the mechanism of CD ET-1 KO-mediated alterations in AVP responsiveness.
CD-derived ET-1 could also modulate water reabsorption through paracrine effects. Adjacent medullary interstitial cells express ET receptors (9) but do not synthesize ET-1 (43). ET-1 elicits cultured medullary interstitial cell NO production (47), which could conceivably act on neighboring CD to inhibit water reabsorption. In addition, medullary CD increases in ET-1 release might lead to augmented medullary blood flow (4, 11), thereby washing out the medullary gradient and facilitating water excretion.
The factors responsible for augmenting CD ET-1 production within a few hours of water loading are unknown. Hypertonicity has been found to reduce cultured CD ET-1 production (20, 33, 38), although this has not been consistently noted (26, 44). Volume depletion, which increases medullary tonicity, decreases renal medullary ET-1 content, whereas volume expansion increases medullary ET-1 levels (20). Another interesting possibility is that increased tubule fluid flow rate may regulate CD ET-1 production. Cai et al. (5) reported that shear stress increases all three NO synthase isoforms in cultured IMCD; the possibility is raised that ET-1 could also be increased and may, at least in part, lead to increased NO production. Given that the IMCD is the major nephron source of ET-1 and that the IMCD is a major site of AVP-stimulated water reabsorption, clarification of this regulatory system is of substantial importance.
In summary, the present studies demonstrate that CD-derived ET-1 is involved in the acute diuretic response to water loading and chronically modulates CD AVP responsiveness. In contrast, CD-derived ET-1 is not necessary for a maximal diuretic response to water loading nor does it play a significant role in mediating AVP escape. Further studies are needed on the mechanisms by which CD-derived ET-1 is regulated and the mechanisms by which it regulates renal water excretion.
GRANTS
This research was funded in part by National Institutes of Health Grants DK-59047 and DK-58953 to D. E. Kohan.
DISCLOSURES
There is no conflict of interest for these studies.
FOOTNOTES
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.
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