Adenylyl cyclase 6 deficiency ameliorates polycystic kidney disease.

cAMP is an important mediator of cystogenesis in polycystic kidney disease (PKD). Several adenylyl cyclase (AC) isoforms could mediate cAMP accumulation in PKD, and identification of a specific pathogenic AC isoform is of therapeutic interest. We investigated the role of AC6 in a mouse model of PKD that is homozygous for the loxP-flanked PKD1 gene and heterozygous for an aquaporin-2-Cre recombinase transgene to achieve collecting duct-specific gene targeting. Collecting duct-specific knockout of polycystin-1 caused massive renal cyst formation, kidney enlargement, and severe kidney failure, with a mean survival time of 2 months. In contrast, coincident collecting duct-specific knockout of polycystin-1 and AC6 (also homozygous for the floxed ADCY6 gene) markedly decreased kidney size and cystogenesis, improved renal function, reduced activation of the B-Raf/ERK/MEK pathway, and greatly increased survival. Absence of collecting duct AC6 did not alter urinary cAMP excretion or kidney cAMP concentration. In conclusion, AC6 is a key mediator of cyst formation and renal injury in a model of PKD.

Na delivery and ENaC mediate flow regulation of collecting duct endothelin-1 production.

Collecting duct (CD) endothelin-1 (ET-1) is an important autocrine inhibitor of Na and water transport. CD ET-1 production is stimulated by extracellular fluid volume expansion and tubule fluid flow, suggesting a mechanism coupling CD Na delivery and ET-1 synthesis. A mouse cortical CD cell line, mpkCCDc14, was subjected to static or flow conditions for 2 h at 2 dyn/cm(2), followed by determination of ET-1 mRNA content. Flow with 300 mosmol/l NaCl increased ET-1 mRNA to 65% above that observed under static conditions. Increasing perfusate osmolarity to 450 mosmol/l with NaCl or Na acetate increased ET-1 mRNA to ∼184% compared with no flow, which was not observed when osmolarity was increased using mannitol or urea. Reducing Na concentration to 150 mosmol/l while maintaining total osmolarity at 300 mosmol/l with urea or mannitol decreased the flow response. Inhibition of epithelial Na channel (ENaC) with amiloride or benzamil abolished the flow response, suggesting involvement of ENaC in flow-regulated ET-1 synthesis. Aldosterone almost doubled the flow response. Since Ca(2+) enhances CD ET-1 production, the involvement of plasma membrane and mitochondrial Na/Ca(2+) exchangers (NCX) was assessed. SEA0400 and KB-R7943, plasma membrane NCX inhibitors, did not affect the flow response. However, CGP37157, a mitochondrial NCX inhibitor, abolished the response. In summary, the current study indicates that increased Na delivery, leading to ENaC-mediated Na entry and mitochondrial NCX activity, is involved in flow-stimulated CD ET-1 synthesis. This constitutes the first report of either ENaC or mitochondrial NCX regulation of an autocrine factor in any biologic system.

Collecting duct-specific knockout of adenylyl cyclase type VI causes a urinary concentration defect in mice.

Collecting duct (CD) adenylyl cyclase VI (AC6) has been implicated in arginine vasopressin (AVP)-stimulated renal water reabsorption. To evaluate the role of CD-derived AC6 in regulating water homeostasis, mice were generated with CD-specific knockout (KO) of AC6 using the Cre/loxP system. CD AC6 KO and controls were studied under normal water intake, chronically water loaded, or water deprived; all of these conditions were repeated in the presence of continuous administration of 1-desamino-8-d-arginine vasopressin (DDAVP). During normal water intake or after water deprivation, urine osmolality (U(osm)) was reduced in CD AC6 KO animals vs. controls. Similarly, U(osm) was decreased in CD AC6 KO mice vs. controls after water deprivation+DDAVP administration. Pair-fed (with controls) CD AC6 KO mice also had lower urine osmolality vs. controls. There were no detectable differences between KO and control animals in fluid intake or urine volume under any conditions. CD AC6 KO mice did not have altered plasma AVP levels vs. controls. AVP-stimulated cAMP accumulation was reduced in acutely isolated inner medullary CD (IMCD) from CD A6 KO vs. controls. Medullary aquaporin-2 (AQP2) protein expression was lower in CD AC6 KO mice vs. controls. There were no differences in urinary urea excretion or IMCD UT-A1 expression; however, IMCD UT-A3 expression was reduced in CD AC6 KO mice vs. controls. In summary, AC6 in the CD regulates renal water excretion, most likely through control of AVP-stimulated cAMP accumulation and AQP2.

Disruption of the endothelin A receptor in the nephron causes mild fluid volume expansion.

BACKGROUND: Endothelin, via endothelin A receptors (ETA), exerts multiple pathologic effects that contribute to disease pathogenesis throughout the body. ETA antagonists ameliorate many experimental diseases and have been extensively utilized in clinical trials. The utility of ETA blockers has been greatly limited, however, by fluid retention, sometimes leading to heart failure or death. To begin to examine this issue, the effect of genetic disruption of ETA in the nephron on blood pressure and salt handling was determined. METHODS: Mice were generated with doxycycline-inducible nephron-specific ETA deletion using Pax8-rtTA and LC-1 transgenes on the background of homozygous loxP-flanked ETA alleles. Arterial pressure, Na metabolism and measures of body fluid volume status (hematocrit and impedance plethysmography) were assessed. RESULTS: Absence of nephron ETA did not alter arterial pressure whether mice were ingesting a normal or high Na diet. Nephron ETA disruption did not detectably affect 24 hr Na excretion or urine volume regardless of Na intake. However, mice with nephron ETA knockout that were fed a high Na diet had mild fluid retention as evidenced by an increase in body weight and a fall in hematocrit. CONCLUSIONS: Genetic deletion of nephron ETA causes very modest fluid retention that does not alter arterial pressure. Nephron ETA, under normal conditions, likely do not play a major role in regulation of Na excretion or systemic hemodynamics.

Identification of two nuclear factor of activated T-cells (NFAT)-response elements in the 5'-upstream regulatory region of the ET-1 promoter.

Collecting duct-derived ET-1 regulates salt excretion and blood pressure. We have reported the presence of an inner medullary collecting duct (IMCD)-specific enhancer region in the 5'-upstream ET-1 promoter (Strait, K. A., Stricklett, P. K., Kohan, J. L., Miller, M. B., and Kohan, D. E. (2007) Am. J. Physiol. Renal Physiol. 293, F601-F606). The current studies provide further characterization of the ET-1 5'-upstream distal promoter to identify the IMCD-specific enhancer elements. Deletion studies identified two regions of the 5'-upstream ET-1 promoter, -1725 to -1319 bp and -1319 to -1026 bp, which were required for maximal promoter activity in transfected rat IMCD cells. Transcription factor binding site analysis of these regions identified two consensus nuclear factor of activated T-cells (NFAT) binding sites at -1263 and -1563. EMSA analysis using nuclear extracts from IMCD cells showed that both the -1263 and the -1563 NFAT sites in the ET-1 distal promoter competed for NFAT binding to previously identified NFAT sites in the IL-2 and TNF genes. Gel supershift analysis showed that each of the NFAT binding sites in the ET-1 promoter bound NFAT proteins derived from IMCD nuclear extracts, but they selectively bound different NFAT isoforms; ET-1263 bound NFATc1, whereas ET-1563 bound NFATc3. Site-directed mutagenesis of either the ET-1263 or the ET-1563 sites prevented NFAT binding and reduced ET-1 promoter activity. Thus, NFAT appears to be an important regulator of ET-1 transcription in IMCD cells, and thus, it may play a role in controlling blood pressure through ET-1 regulation of renal salt excretion.

Flow regulation of collecting duct endothelin-1 production.

Collecting duct (CD) endothelin-1 (ET-1) is an important autocrine inhibitor of CD Na(+) reabsorption. Salt loading is thought to increase CD ET-1 production; however, definitive evidence of this, as well as understanding of the mechanisms transducing this effect, is lacking. Tubule fluid flow increases in response to Na(+) loading; hence, we studied flow modulation of CD ET-1 production. Three days of a high-salt diet increased mouse and rat inner medullary CD (IMCD) ET-1 mRNA expression. Acute furosemide infusion increased urinary ET-1 excretion in anesthetized rats. Primary cultures of mouse or rat IMCD detached in response to flow using a closed perfusion chamber, consequently a CD cell line (mpkCCDcl4) was examined. Flow increased ET-1 mRNA at shear stress rates exceeding 1 dyne/cm(2), with the maximal effect seen between 2 and 10 dyne/cm(2). Induction of ET-1 mRNA was first evident after 1 h, and most apparent after 2 h, of flow. Inhibition of calmodulin or dihydropyridine-sensitive Ca(2+) channels did not alter the flow response; however, chelation of intracellular Ca(2+) or removal of extracellular Ca(2+) largely prevented flow-stimulated ET-1 mRNA accumulation. Downregulation of protein kinase C (PKC) using phorbol 12-myristate 13-acetate, or PKC inhibition with calphostin C, markedly reduced flow-stimulated ET-1 mRNA levels. Flow-stimulated ET-1 mRNA accumulation was abolished by inhibition of phospholipase C (PLC). Taken together, these data indicate that flow increases CD ET-1 production and this is dependent on extracellular and intracellular Ca(2+), PKC, and PLC. These studies suggest a novel pathway for coupling alterations in extracellular fluid volume to CD ET-1 production and ultimately control of CD Na(+) reabsorption.

Characterization of vasopressin-responsive collecting duct adenylyl cyclases in the mouse.

Little is known about collecting duct adenylyl cyclase (AC) isoforms or regulation in the mouse. We performed RT-PCR for AC isoforms 1-9 in microdissected cortical (CCD) and outer medullary (OMCD) and acutely isolated inner medullary (IMCD) collecting duct. All collecting duct regions contained AC3, AC4, and AC6 mRNA, while CCD and OMCD, but not IMCD, also contained AC5 mRNA. Acutely isolated IMCD expressed AC3, AC4, and AC6 proteins by Western blot analysis. The mIMCD3 cell line expressed AC2, AC3, AC4, AC5, and AC6 mRNA; M-1 CCD cells expressed AC2, 3, 4, and 6, while mpkCCD cell lines contained AC3, AC4, and AC6 mRNA. AVP stimulated cAMP accumulation in acutely isolated mouse IMCD; this was reduced by chelation of extracellular calcium (EGTA) and almost completely abolished by blockade of calmodulin (W-7). Blockade of calmodulin kinase with KN-93 or endoplasmic reticulum calcium ATPase (thapsigargin) also reduced the AVP response. A similar inhibitory effect of W-7, KN-93, and thapsigargin was seen on forskolin-stimulated cAMP content in acutely isolated mouse IMCD. These three agents had the same pattern of blockade of AVP- or forskolin-stimulated AC activity in acutely isolated rat IMCD. AVP responsiveness in primary cultures of mouse IMCD was also reduced by W-7, KN-93, and thapsigargin. Small interfering RNA (siRNA) designed to knock down AC3 or AC6 in primary cultured mouse IMCD significantly reduced AVP-stimulated cAMP accumulation. Together, these data are consistent with a role of AC3 and AC6 in the activation of mouse collecting duct by AVP.

Inactivation of Pkd1 in principal cells causes a more severe cystic kidney disease than in intercalated cells.

Renal cysts in autosomal dominant polycystic kidney disease arise from cells throughout the nephron, but there is an uncertainty as to whether both the intercalated cells (ICs) and principal cells (PCs) within the collecting duct give rise to cysts. To determine this, we crossed mice containing loxP sites within introns 1 and 4 of the Pkd1 gene with transgenic mice expressing Cre recombinase under control of the aquaporin-2 promoter or the B1 subunit of the proton ATPase promoter, thereby generating PC- or IC-specific knockout of Pkd1, respectively. Mice, that had Pkd1 deleted in the PCs, developed progressive cystic kidney disease evident during the first postnatal week and had an average lifespan of 8.2 weeks. There was no change in the cellular cAMP content or membrane aquaporin-2 expression in their kidneys. Cysts were present in the cortex and outer medulla but were absent in the papilla. Mice in which PKd1 was knocked out in the ICs had a very mild cystic phenotype as late as 13 weeks of age, limited to 1-2 cysts and confined to the outer rim of the kidney cortex. These mice lived to at least 1.5 years of age without evidence of early mortality. Our findings suggest that PCs are more important than ICs for cyst formation in polycystic kidney disease.

Altered collecting duct adenylyl cyclase content in collecting duct endothelin-1 knockout mice.

BACKGROUND: Endothelin-1 (ET-1) inhibition of vasopressin (AVP)-stimulated water reabsorption by the inner medullary collecting duct (IMCD) is associated with reduced cAMP accumulation. To determine the effect of ET-1 deficiency, AVP-stimulated cAMP responsiveness was assessed in IMCD from mice with collecting duct-specific deletion of ET-1 (CD ET-1 KO) and from control animals. METHODS: Cyclic AMP production, adenylyl cyclase (AC) mRNA, and AC protein were measured in acutely isolated IMCD. RESULTS: CD ET-1 KO IMCD had enhanced AVP-stimulated cAMP accumulation. Inhibition of calcium-stimulated AC using BAPTA did not prevent enhanced AVP responsiveness in CD ET-1 KO IMCD. Factors known to be modified by ET-1, including nitric oxide, cyclooxygenase metabolites, and superoxide did not affect the increased AVP responsiveness of CD ET-1 KO IMCD. Differential V2 receptor or G-protein activity was not involved since CD ET-1 KO IMCD had increased cAMP accumulation in response to forskolin and/or cholera toxin. CD ET-1 KO did not affect mRNA or protein levels of AC3, one of the major known collecting duct AC isoforms. However, the other known major collecting duct AC isoform (AC5/6) did have increased protein levels in CD ET-1 KO IMCD, although AC5 (weak signal) and 6 mRNA levels were unchanged. CONCLUSION: ET-1 deficiency increases IMCD AC5/6 content, an effect that may synergize with acute ET-1 inhibition of AVP-stimulated cAMP accumulation.

Histone deacetylase inhibitors induce G2-checkpoint arrest and apoptosis in cisplatinum-resistant ovarian cancer cells associated with overexpression of the Bcl-2-related protein Bad.

Trichostatin A produces predominantly G(1) cell-cycle blockade and differentiation of the cisplatinum-sensitive A2780 ovarian cancer cell line. Given the propensity of ovarian tumors to become resistant to cisplatinum, often leading to cross-resistance to other agents, we have extended these observations by examining how the emergence of resistant phenotypes in A2780 cells affects the actions of histone deacetylase (HDAC) inhibitors. Trichostatin A exposure (100 ng/mL, 24 hours) induced ultrastructural differentiation of the "intrinsically" cisplatinum-resistant A2780-9M subline, with the reappearance of intercellular junctions and lumina containing primitive microvilli. Similar trichostatin A exposure in the acquired resistance A2780CP cells produced minimal differentiation consisting of occasional weak intercellular junctions. Independent of the differences in trichostatin A-induced differentiation, in both resistant sublines trichostatin A produced a similar reduction in cell viability, by >90%, within 5 days of treatment. Diminished viability in both A2780-9M and CP cells was associated with the absence of cell cycle arrest in G1, resulting in predominant G2-checkpoint arrest accompanied by a 10- to 20-fold increase in Annexin V binding and the reemergence of apoptosis. Similar cell cycle arrests and apoptosis were also observed using other HDAC inhibitors and in other resistant ovarian cancer cell lines (OVCAR-3 and SK-OV-3). Trichostatin A-induced apoptosis in resistant cells is in sharp contrast to its effects on the parental cisplatinum-sensitive A2780 and normal MRC-5 fibroblast cell lines (predominant cycle arrest in G1 with no detectable apoptosis). Western immunoblot analysis indicated trichostatin A triggers apoptosis in resistant ovarian cancer cells via p53-independent activation of the intrinsic "mitochondrial" pathway, commensurate with induction of the Bcl-2-related protein Bad. These results suggest cisplatinum resistance alters the effects of HDAC inhibition through a shift in cell cycle arrest from the G1 to the G2 checkpoint and reactivation of the intrinsic mitochondrial apoptotic cascade.

Cell type-dependent regulation of hMLH1 promoter activity is influenced by the presence of multiple redundant elements.

Immunohistochemical analysis confirmed the presence of MLH1 protein in A2780 ovarian cancer cells and its absence in this same cell line on acquired resistance to cisplatinum (A2780/CP). Transfection of a -1781-bp hMLH1 promoter construct into either A2780 or A2780/CP cells produced similar (30-fold) induction of luciferase, an indication that the transcriptional machinery for hMLH1 expression remains intact. hMLH1-luciferase activity was also unaffected by re-expression of hMLH1 following treatment of A2780/CP cells with the methylase inhibitor 2'-deoxy-5-azacytidine. Serial 5'-deletion studies of the hMLH1 promoter region in ovarian cancer cells localized transcriptional enhancers to a region (-250 to -151 bp) that excludes the previously identified CCAAT element (-282) active in HeLa cells. When these same deletion constructs were transfected into HeLa cells, deletion of the CCAAT-containing region caused a significant loss of promoter activity, an indication of cell-specific use of enhancer elements. Finally, a series of internal deletion and linker mutation studies of the -250 to -151 bp ovarian enhancer region revealed that the hMLH1 promoter contains multiple redundant enhancer elements capable of independent promoter activation and may explain the association of this region with methylation silencing of hMLH1.

Cell cycle blockade and differentiation of ovarian cancer cells by the histone deacetylase inhibitor trichostatin A are associated with changes in p21, Rb, and Id proteins.

Inhibitors of histone deacetylase activity are emerging as a potentially important new class of anticancer agents. In the current studies, exposing A2780 ovarian cancer cells to the histone deacetylase inhibitor trichostatin A (TSA) produced a marked change in cellular morphology, proliferation, and differentiation. Within 24 h of TSA treatment, there was a morphological transformation of the cells, with increased cytoplasm, a more epithelial-like columnar appearance, and the emergence of distinct cellular boundaries. Commensurate with the morphological transformation, TSA also inhibited cell proliferation; cells treated with TSA for 72 h increased to 110% of the initial cell numbers versus control cell numbers of 622%, with a corresponding reduction in mitotic activity and a flow cytometry S-phase fraction of 3.9% in TSA-treated cells versus 28.8% for control. TSA also induced epithelial-like differentiation with increased cytokeratin expression from 2% of controls to 22-25% of TSA-treated cells and the reappearance of intercellular plasma membrane junctions and primitive microvilli. Immunocytochemical analyses indicate the molecular mechanism underlying the actions of TSA on A2780 cell cycle progression and differentiation involves reexpression of the CDK inhibitor p21. Elevated levels of p21, in TSA-treated cells, were associated with a reduction in the phosphorylation of the cell cycle regulator retinoblastoma protein (Rb). TSA also caused a decrease in the helix-loop-helix inhibitor of differentiation/DNA binding protein Id1, with no change in Id2 levels. In conclusion, the observed TSA-induced changes in p21, Rb, and Id1 are consistent with cell cycle senescence and differentiation of A2780 ovarian cancer cells.

Novel regulation of endothelin-1 promoter activity by protein kinase C.

Endothelin-1 (ET-1) is produced in unusually large amounts by the renal collecting duct and acts locally to control renal salt and water excretion and arterial pressure; disorders of collecting duct ET-1 activity can cause marked hypertension. The mechanisms regulating collecting duct ET-1 synthesis are, however, poorly understood. In this study, we investigated the role of protein kinase C (PKC), a known regulator of ET-1 production in endothelial cells, in (1) the control of collecting duct ET-1 production; and (2) the modulation of ET-1 promoter region activity. Cultured rat inner medullary collecting duct (IMCD) cells were studied. Calphostin C, a PKC inhibitor, greatly reduced IMCD ET-1 release. Sustained exposure to phorbol myristate acetate (PMA) also decreased ET-1 secretion. PKC inhibition decreased steady-state ET-1 mRNA content. A brief exposure (15 min) to PMA augmented ET-1 mRNA levels, while prolonged PMA exposure (120 min) reduced ET-1 mRNA content, PKC inhibition did not affect ET-1 mRNA stability. Transfection of ET-1 promoter-luciferase reporter constructs into IMCD cells demonstrated that PKC inhibition reduced activity of only the larger promoter fragments (containing at least 1,725 bp 5' to the ET-1 gene transcription start site). Mutation of a previously identified AP-1 site at -186 in the ET-1 promoter greatly reduced activity of transfected ET-1 promoter-reporter constructs (containing 366 or 1,725 bp 5' to the transcription start site); however, this region appears not to be regulated by PKC in IMCD cells. In summary, PKC stimulates collecting duct ET-1 synthesis via transcriptional activation of the ET-1 promoter. Such transcriptional activation occurs at a heretofore undescribed PKC-regulated region of the ET-1 promoter.

Novel mechanism for regulation of endothelin synthesis: role of extracellular pH.

BACKGROUND/AIMS: Several studies suggest that pH may be an important regulator of renal collecting duct endothelin-1 (ET-1) synthesis; the current study was undertaken to examine this interaction. METHODS: Rat inner medullary collecting duct (IMCD) cells were exposed to varying extracellular pH and ET-1 biosynthetic pathways assessed. RESULTS: Exposure of IMCD to varying pH revealed a strong direct correlation between media pH and both mature ET-1 and Big ET-1 release. A direct correlation also existed between cellular Big ET-1 content and pH. Varying media pH did not alter ET-1 mRNA content nor did it affect activity of a transfected 3.2 kb ET-1 promoter. Media pH did not affect ET-1 degradation nor did it alter IMCD cell total protein synthesis ((3)H-leucine incorporation). Attempts to measure ET-1 mRNA translation rate using His-Tag labeled ET-1 cDNA were not successful. CONCLUSION: pH regulates IMCD ET-1 production through regulation of either mRNA translation or preproET-1 cleavage. This represents a novel mechanism for regulation of ET-1 synthesis.

Calcium regulation of endothelin-1 synthesis in rat inner medullary collecting duct.

Collecting duct-derived endothelin-1 (ET-1) reduces blood pressure and inhibits Na and water reabsorption. Collecting duct ET-1 production is increased by volume expansion; however, the mechanism by which this occurs is unknown. We hypothesized that intracellular calcium, which is likely to be increased by volume expansion, regulates collecting duct ET-1 synthesis. Rat inner medullary collecting ducts (IMCD) were studied in primary culture. ET-1 release was decreased by 50-70% after chelation of intracellular calcium (BAPTA) or inhibition of CaM (W7) or CaMK (KN-93). These agents reduced ET-1 mRNA to a similar degree. CaM inhibition did not affect ET-1 mRNA stability. Transfection of IMCD with rat ET-1 promoter-luciferase constructs revealed maximal activity within 1.7 kb 5' to the transcription start site; 5, 20, 35, and 90% of this activity were in the 0.08-, 0.37-, 1.0-, and 3.0-kb promoter regions, respectively. W7 markedly inhibited activity of the 3.0-kb but not 0.37- or 1.0-kb promoter regions. In contrast, W7 did not affect ET-1 release by rat aortic endothelial cells. Furthermore, transfected endothelial cells had maximal activity in the 0.37-kb region (as compared with the 1.7- and 3.0-kb regions), whereas W-7 had no effect on the activity of any of these promoter regions. In summary, IMCD ET-1 synthesis is regulated by calcium/CaM/CaMK-dependent pathways. The calcium/CaM-sensitive pathway is active in IMCD, but not endothelial cells. This suggests that IMCD-specific enhancer elements exist within the ET-1 promoter that confer unique calcium responsiveness.

Role of prostaglandins in collecting duct-derived endothelin-1 regulation of blood pressure and water excretion.

Collecting duct (CD)-derived endothelin-1 (ET-1) exerts natriuretic, diuretic, and hypotensive effects. In vitro studies have implicated cyclooxygenase (COX) metabolites, and particularly PGE(2), as important mediators of CD ET-1 effects. However, it is unknown whether PGE(2) mediates CD-derived ET-1 actions in vivo. To test this, CD ET-1 knockout (KO) and control mice were studied. During normal salt and water intake, urinary PGE(2) excretion was unexpectedly increased in CD ET-1 KO mice compared with controls. Salt loading markedly increased urinary PGE(2) excretion in both groups of mice; however, the levels remained relatively higher in KO animals. Acutely isolated inner medullary collecting duct (IMCD) from KO mice also had increased PGE(2) production. The increased IMCD PGE(2) was COX-2 dependent, since NS-398 blocked all PGE(2) production. However, increased CD ET-1 KO COX-2 protein or mRNA could not be detected in inner medulla or IMCD, respectively. Inner medullary COX-1 mRNA and protein levels and IMCD COX-1 mRNA levels were unaffected by Na intake or CD ET-1 KO. KO mice on a normal or high-Na diet had elevated blood pressure compared with controls; this difference was not altered by indomethacin or NS-398 treatment. However, indomethacin or NS-398 did increase urine osmolality and reduce urine volume in KO, but not control, animals. In summary, IMCD COX-2-dependent PGE(2) production is increased in CD ET-1 KO mice, indicating that CD-derived ET-1 is not a primary regulator of IMCD PGE(2). Furthermore, the increased PGE(2) in CD ET-1 KO mice partly compensates for loss of ET-1 with respect to maintaining urinary water excretion, but not in blood pressure control.