Polyhydramnios, Transient Antenatal Bartter's Syndrome, and MAGED2 Mutations.

BACKGROUND: Three pregnancies with male offspring in one family were complicated by severe polyhydramnios and prematurity. One fetus died; the other two had transient massive salt-wasting and polyuria reminiscent of antenatal Bartter's syndrome. METHODS: To uncover the molecular cause of this possibly X-linked disease, we performed whole-exome sequencing of DNA from two members of the index family and targeted gene analysis of other members of this family and of six additional families with affected male fetuses. We also evaluated a series of women with idiopathic polyhydramnios who were pregnant with male fetuses. We performed immunohistochemical analysis, knockdown and overexpression experiments, and protein-protein interaction studies. RESULTS: We identified a mutation in MAGED2 in each of the 13 infants in our analysis who had transient antenatal Bartter's syndrome. MAGED2 encodes melanoma-associated antigen D2 (MAGE-D2) and maps to the X chromosome. We also identified two different MAGED2 mutations in two families with idiopathic polyhydramnios. Four patients died perinatally, and 11 survived. The initial presentation was more severe than in known types of antenatal Bartter's syndrome, as reflected by an earlier onset of polyhydramnios and labor. All symptoms disappeared spontaneously during follow-up in the infants who survived. We showed that MAGE-D2 affects the expression and function of the sodium chloride cotransporters NKCC2 and NCC (key components of salt reabsorption in the distal renal tubule), possibly through adenylate cyclase and cyclic AMP signaling and a cytoplasmic heat-shock protein. CONCLUSIONS: We found that MAGED2 mutations caused X-linked polyhydramnios with prematurity and a severe but transient form of antenatal Bartter's syndrome. MAGE-D2 is essential for fetal renal salt reabsorption, amniotic fluid homeostasis, and the maintenance of pregnancy. (Funded by the University of Groningen and others.).

Bartter's and Gitelman's syndrome.

PURPOSE OF REVIEW: The clinical presentations of Bartter's syndrome and Gitelman's syndrome will be reviewed including two most recently described hypokalemic salt-losing tubulopathies. By taking the quite heterogeneous presentations and the apparently different pathophysiologies as the basis, the applicability of the physiologic classification has been tested. RECENT FINDINGS: According to the physiologic approach, salt-losing tubulopathies can be divided into two major groups (with completely different tubular defects): first, disorders of the thick ascending limb of Henle's loop (loop disorders); second, disorders of the distal convolute tubule (DCT disorders). A combination of these two groups with complety different tubular defects will finally lead to a third group: the combined loop/DCT disorders. On the basis of pharmacologic tests (pharmacotyping), it appears that the Bartter's syndrome V belongs to the DCT group, whereas the most recently described transient antenatal Bartter's syndrome best fits in the group with the loop and DCT combination.Besides secondary hyperaldosteronism, loop disorders present a whole spectrum of (secondary) pathophysiologic characteristics with significant diagnostic and therapeutic impact, such as polyhydramnios, hyperprostaglandinuria, nephrogenic diabetes insipidus, and nephrocalcinosis. Recent reports indicate that neonatal hyperparathyroidism has also to be added to the clinical presentation of isolated loop disorders. SUMMARY: As long as gene therapy is not available, the overall therapeutic management follows the clinical presentation, which leads to the underlying pathophysiology of renal salt wasting. Thus, when dealing with Bartter's syndrome and Gitelman's syndrome, the correct physiologic and pharmacologic characterization appears to be essential for a sound diagnostic and therapeutic patient management.

Pathophysiology and clinical presentations of salt-losing tubulopathies.

At least three renal tubular segments are involved in the pathophysiology of salt-losing tubulopathies (SLTs). Whether the pathogenesis starts either in the thick ascending limb of the loop of Henle (TAL) or in the distal convoluted tubule (DCT), it is the function of the downstream-localized aldosterone sensitive distal tubule (ASDT) to contribute to the adaptation process. In isolated TAL defects (loop disorders) ASDT adaptation is supported by upregulation of DCT, whereas in DCT disorders the ASDT is complemented by upregulation of TAL function. This upregulation has a major impact on the clinical presentation of SLT patients. Taking into account both the symptoms and signs of primary tubular defect and of the secondary reactions of adaptation, a clinical diagnosis can be made that eventually leads to an appropriate therapy. In addition to salt wasting, as occurs in all SLTs, characteristic features of loop disorders are hypo- or isosthenuric polyuria and hypercalciuria, whereas characteristics of DCT disorders are hypokalemia and (symptomatic) hypomagnesemia. In both SLT categories, replacement of urinary losses is the primary goal of treatment. In loop disorders COX inhibitors are also recommended to mitigate polyuria, and in DCT disorders magnesium supplementation is essential for effective treatment. Of note, the combination of a salt- and potassium-rich diet together with an adequate fluid intake is always the basis of long-term treatment in all SLTs.

Hypomagnesemia with secondary hypocalcemia is caused by mutations in TRPM6, a new member of the TRPM gene family.

Magnesium is an essential ion involved in many biochemical and physiological processes. Homeostasis of magnesium levels is tightly regulated and depends on the balance between intestinal absorption and renal excretion. However, little is known about specific proteins mediating transepithelial magnesium transport. Using a positional candidate gene approach, we identified mutations in TRPM6 (also known as CHAK2), encoding TRPM6, in autosomal-recessive hypomagnesemia with secondary hypocalcemia (HSH, OMIM 602014), previously mapped to chromosome 9q22 (ref. 3). The TRPM6 protein is a new member of the long transient receptor potential channel (TRPM) family and is highly similar to TRPM7 (also known as TRP-PLIK), a bifunctional protein that combines calcium- and magnesium-permeable cation channel properties with protein kinase activity. TRPM6 is expressed in intestinal epithelia and kidney tubules. These findings indicate that TRPM6 is crucial for magnesium homeostasis and implicate a TRPM family member in human disease.

Bartter- and Gitelman-like syndromes: salt-losing tubulopathies with loop or DCT defects.

Salt-losing tubulopathies with secondary hyperaldosteronism (SLT) comprise a set of well-defined inherited tubular disorders. Two segments along the distal nephron are primarily involved in the pathogenesis of SLTs: the thick ascending limb of Henle's loop, and the distal convoluted tubule (DCT). The functions of these pre- and postmacula densa segments are quite distinct, and this has a major impact on the clinical presentation of loop and DCT disorders - the Bartter- and Gitelman-like syndromes. Defects in the water-impermeable thick ascending limb, with its greater salt reabsorption capacity, lead to major salt and water losses similar to the effect of loop diuretics. In contrast, defects in the DCT, with its minor capacity of salt reabsorption and its crucial role in fine-tuning of urinary calcium and magnesium excretion, provoke more chronic solute imbalances similar to the effects of chronic treatment with thiazides. The most severe disorder is a combination of a loop and DCT disorder similar to the enhanced diuretic effect of a co-medication of loop diuretics with thiazides. Besides salt and water supplementation, prostaglandin E2-synthase inhibition is the most effective therapeutic option in polyuric loop disorders (e.g., pure furosemide and mixed furosemide-amiloride type), especially in preterm infants with severe volume depletion. In DCT disorders (e.g., pure thiazide and mixed thiazide-furosemide type), renin-angiotensin-aldosterone system (RAAS) blockers might be indicated after salt, potassium, and magnesium supplementation are deemed insufficient. It appears that in most patients with SLT, a combination of solute supplementation with some drug treatment (e.g., indomethacin) is needed for a lifetime.

Basics and dynamics of neonatal and pediatric pharmacology.

Understanding the role of ontogeny in the disposition and actions of medicines is the most fundamental prerequisite for safe and effective pharmacotherapeutics in the pediatric population. The maturational process represents a continuum of growth, differentiation, and development, which extends from the very small preterm newborn infant through childhood, adolescence, and to young adulthood. Developmental changes in physiology and, consequently, in pharmacology influence the efficacy, toxicity, and dosing regimen of medicines. Relevant periods of development are characterized by changes in body composition and proportion, developmental changes of physiology with pathophysiology, exposure to unique safety hazards, changes in drug disposition by major organs of metabolism and elimination, ontogeny of drug targets (e.g., enzymes, transporters, receptors, and channels), and environmental influences. These developmental components that result in critical windows of development of immature organ systems that may lead to permanent effects later in life interact in a complex, nonlinear fashion. The ontogeny of these physiologic processes provides the key to understanding the added dimension of development that defines the essential differences between children and adults. A basic understanding of the developmental dynamics in pediatric pharmacology is also essential to delineating the future directions and priority areas of pediatric drug research and development.

Loop disorders: insights derived from defined genotypes.

Great progress has been made in the last 15 years in the characterization and the pathophysiological understanding of renal salt and water wasting associated with inherited disorders of the thick ascending limb (TAL) of Henle's loop, the loop disorders. Besides careful clinical observations and innovative physiological concepts, molecular genetics have made this progress possible. So far, mutations in five different genes may be responsible for the loop disorders. These gene products are as follows: NKCC2 symporter, ROMK, ClC-Ka, ClC-Kb, and barttin, a ß-subunit to both chloride channels. The key symptoms, such as polyhydramnios secondary to fetal polyuria, postnatal volume depletion with hypotension, iso- or hyposthenuria, hyperprostaglandinuria and hypercalciuria followed by hypokalemic alkalosis secondary to hyperaldosteronism, are typical features of loop disorders that are restricted to TAL, such as in disorders with NKCC2 and ROMK mutations. However, transient perinatal hyperkalemia in infants with ROMK mutations suggests an additional function of ROMK for K secretion in the cortical collecting duct. The extremely rare human ClC-Kamutation has only been described in combination with ClC-Kb mutations. Similar to barttin mutations, this double knockout of transepithelial salt transport in TAL and in distal convoluted tubule (DCT) leads to a severe loop disorder with deafness. In contrast, the isolated ClC-Kb mutation predominantly appears as an incomplete loop disorder with features similar to an isolated DCT defect, because ClC-Kb function in TAL can in part be compensated by ClC-Ka. This compensation does not exist in DCT. Besides these defined genotypes, the type and the severity of mutation as well as the onset and quality of medical care are important determinants for the patients' outcome. Considering a few variables, such as transient hyperkalemia, disease onset beyond neonatal period, profound hypochloremia and hypokalemia, or congenital hearing loss, might be helpful to guide genetic testing efficiently.

An improved terminology and classification of Bartter-like syndromes.

This Review outlines a terminology and classification of Bartter-like syndromes that is based on the underlying causes of these inherited salt-losing tubulopathies and is, therefore, more clinically relevant than the classical definition. Three major types of salt-losing tubulopathy can be defined: distal convoluted tubule dysfunction leading to hypokalemia (currently known as Gitelman or Bartter syndrome), the more-severe condition of polyuric loop dysfunction (often referred to as antenatal Bartter or hyperprostaglandin E syndrome), and the most-severe condition of combined loop and distal convoluted tubule dysfunction (antenatal Bartter or hyperprostaglandin E syndrome with sensorineural deafness). These three subtypes can each be further subdivided according to the identity of the defective ion transporter or channel: the sodium-chloride cotransporter NCCT or the chloride channel ClC-Kb in distal convoluted tubule dysfunction; the sodium-potassium-chloride cotransporter NKCC2 or the renal outer medullary potassium channel in loop dysfunction; and the chloride channels ClC-Ka and ClC-Kb or their beta-subunit Barttin in combined distal convoluted tubule and loop dysfunction. This new classification should help clinicians to better understand the pathophysiology of these syndromes and choose the most appropriate treatment for affected patients, while avoiding potentially harmful diagnostic and therapeutic approaches.

Clinical presentation of genetically defined patients with hypokalemic salt-losing tubulopathies.

PURPOSE: Hypokalemic salt-losing tubulopathies (Bartter-like syndromes) comprise a set of clinically and genetically distinct inherited renal disorders. Mutations in four renal membrane proteins involved in electrolyte reabsorption have been identified in these disorders: the furosemide-sensitive sodium-potassium-chloride cotransporter NKCC2, the potassium channel ROMK, the chloride channel ClC-Kb, and the thiazide-sensitive sodium-chloride cotransporter NCCT. The aim of this study was to characterize the clinical features associated with each mutation in a large cohort of genetically defined patients. PATIENTS AND METHODS: The phenotypic characteristics of 65 patients with molecular defects in NKCC2, ROMK, ClC-Kb, or NCCT were collected retrospectively. RESULTS: ROMK and NKCC2 patients presented with polyhydramnios, nephrocalcinosis, and hypo- or isosthenuria. Hypokalemia was less severe in the ROMK patients compared with the NKCC2 patients. In contrast, NCCT patients had hypocalciuria, hypomagnesemia, and marked hypokalemia. While this dissociation of renal calcium and magnesium handling was also observed in some ClC-Kb patients, a few ClC-Kb patients presented with hypercalciuria and hypo- or isosthenuria. CONCLUSIONS: ROMK, NKCC2, and NCCT mutations usually have uniform clinical presentations, whereas mutations in ClC-Kb occasionally lead to phenotypic overlaps with the NCCT or, less commonly, with the ROMK/NKCC2 cohort. Based on these results, we propose an algorithm for the molecular diagnosis of hypokalemic salt-losing tubulopathies.

Effects of celecoxib and diclofenac on blood pressure, renal function, and vasoactive prostanoids in young and elderly subjects.

Cyclooxygenase (COX) inhibitors are among the most widely used drugs, especially in the elderly. It has been claimed that the new COX-2 inhibitors offer advantages in terms of drug safety. To test this hypothesis, the authors compared in a double-blind, randomized trial the effects of celecoxib (200 mg bid) and diclofenac (75 mg bid) on blood pressure and renal function in two groups (each n = 12) of young (mean age = 32 years) and elderly (mean age = 68 years) normotensive subjects. Changes from baseline in the 24-hour blood pressure profiles, parameters of the renin-angiotensin-aldosterone system, inulin clearance, urinary marker proteins, and eicosanoid excretion were monitored during the treatment period of 2 weeks. Comparison between celecoxib and diclofenac showed no significant difference in minor alterations of blood pressure. During daytime, there was a trend to elevation of mean arterial blood pressure (mmHg) by celecoxib in the elderly of 2.8 (95 % confidence interval [CI) = -2.5 to 8.2) in comparison with the young subjects of -1.3 (95% CI= -3.7 to 1.0); there was also a trend to elevation of mean arterial blood pressure by diclofenac in the elderly of 4.1 (95% CI= -1.2 to 9.4) in comparison with the young subjects of 0.4 (95% CI= -2.4 to 3.2). In both populations, the authors found no significant drug effects on the parameters of the renin-angiotensin-aldosterone system, inulin clearance, and urinary marker proteins. As expected, diclofenac reduced excretion of allprostanoids, whereas celecoxib did not affect production of TxB2 and its metabolites. Neither in young nor in elderly normotensive subjects were blood pressure and renal function significantly affected by a short-term treatment with standard doses of celecoxib and diclofenac. Therefore, normal aging appears not to represent a special risk factor in therapy with these two agents.

Determination of misoprostol free acid in human breast milk and serum by gas chromatography/negative ion chemical ionization tandem mass spectrometry.

To study an expected transition of misoprostol from human blood into breast milk, a novel method for the determination of its active metabolite misoprostol acid (MPA) was developed. MPA was determined in serum and breast milk samples by an isotope dilution assay using gas chromatography/negative ion chemical ionization tandem mass spectrometry (GC/NICI-MS/MS). After addition of (15S)-15-methylprostaglandin E(2) (15-methyl-PGE(2)) as an internal standard, MPA was extracted from both matrices using a reversed-phase cartridge. The prostanoids were derivatized with O-2,3,4,5,6-pentafluorobenzylhydroxylamine hydrochloride (PFBHA) and 2,3,4,5,6-pentafluorobenzyl bromide (PFBB) to the pentafluorobenzyl oxime (PFBO)-pentafluorobenzyl ester (PFB) derivatives. The sample was subjected to thin-layer chromatography with ethyl acetate-hexane (1 : 1 (v/v)) as the developing solvent. The corresponding zone was extracted. After derivatization to the trimethylsilyl ether, MPA was determined by GC/NICI-MS/MS using the [molecule (M) - pentafluorobenzyl (PFB)](-) ([P](-)) ions as precursor in the negative ion chemical ionization mode. The product ions used for quantification were [P - 2TMSOH - C(6)F(5)CH(2)OH](-) (MPA) and [P - 2TMSOH - C(6)F(5)CH(2)OH - CO(2)](-)(15-methyl-PGE(2)), respectively. The limit of quantification for MPA was approximately 1 pg ml(-1) in breast milk and serum samples. The correlation coefficients of the calibration curves for MPA were r > 0.997 in the 0.5-2000 pg ml(-1) range for both tested matrices.

Increased systolic blood pressure with rofecoxib in congenital furosemide-like salt loss.

BACKGROUND: To analyse whether congenital furosemide- or thiazide-like renal salt loss protects against the potential prohypertensive effects of two cyclooxygenase (COX) inhibitors: rofecoxib, a COX-2 selective inhibitor, and indomethacin, an unselective COX-inhibitor. METHODS: In a retrospective analysis, the effects of rofecoxib and indomethacin on blood pressure (bp: transformed into age-independent standard deviation scores (SDS) values), creatinine clearance (CRC), fractional excretion of sodium (FeNa), and renal excretion of systemic prostaglandins were studied in 28 patients with a genetically proven congenital hypokalaemic salt-losing tubulopathy (SLT) (11 female and 17 male, age: 2-25 years), 19 with a furosemide-like SLT, and nine with a thiazide-like SLT. RESULTS: In furosemide-like SLT patients, systolic SDS bp values were significantly higher with rofecoxib (1.03 +/- 0.16 SDS, n = 107) compared with indomethacin (0.56 +/- 0.09 SDS, n = 282; P = 0.007, 95% CI: 0.12-0.8). Without the drugs, systolic SDS bp values were elevated by 0.63 +/- 0.11 SDS, n = 164. Both drugs reduced renin and aldosterone-plasma levels to a similar extent. SDS values were significantly correlated with the excretion of the vasoconstrictor thromboxane (T x B2) (R2: 0.038, P = 0.021, n: 159), but not with CRC or FeNa. Blood pressure was not increased in thiazide-like SLT patients treated with rofecoxib. CONCLUSION: Congenital furosemide-like renal salt loss does not protect against the prohypertensive effects of rofecoxib. The positive correlation between bp SDS values with T x B2 but not with FeNa or CRC point towards an altered vascular homeostasis as one mechanism increasing blood pressure.

Late-onset manifestation of antenatal Bartter syndrome as a result of residual function of the mutated renal Na+-K+-2Cl- co-transporter.

Genetic defects of the Na+-K+-2Cl- (NKCC2) sodium potassium chloride co-transporter result in severe, prenatal-onset renal salt wasting accompanied by polyhydramnios, prematurity, and life-threatening hypovolemia of the neonate (antenatal Bartter syndrome or hyperprostaglandin E syndrome). Herein are described two brothers who presented with hyperuricemia, mild metabolic alkalosis, low serum potassium levels, and bilateral medullary nephrocalcinosis at the ages of 13 and 15 yr. Impaired function of sodium chloride reabsorption along the thick ascending limb of Henle's loop was deduced from a reduced increase in diuresis and urinary chloride excretion upon application of furosemide. Molecular genetic analysis revealed that the brothers were compound heterozygotes for mutations in the SLC12A1 gene coding for the NKCC2 co-transporter. Functional analysis of the mutated rat NKCC2 protein by tracer-flux assays after heterologous expression in Xenopus oocytes revealed significant residual transport activity of the NKCC2 p.F177Y mutant construct in contrast to no activity of the NKCC2-D918fs frameshift mutant construct. However, coexpression of the two mutants was not significantly different from that of NKCC2-F177Y alone or wild type. Membrane expression of NKCC2-F177Y as determined by luminometric surface quantification was not significantly different from wild-type protein, pointing to an intrinsic partial transport defect caused by the p.F177Y mutation. The partial function of NKCC2-F177Y, which is not negatively affected by NKCC2-D918fs, therefore explains a mild and late-onset phenotype and for the first time establishes a mild phenotype-associated SLC12A1 gene mutation.

Perinatal management of a preterm neonate affected by hyperprostaglandin E2 syndrome (HPS).

BACKGROUND: Neonates affected by hyperprostaglandin E(2) syndrome (HPS) present with severe polyuria. Both urinary losses as well as prostaglandin synthesis inhibitors may precipitate acute renal failure (ARF). AIM: Our goal was to maintain euvolaemia by replacement of urinary losses. PATIENT: Our patient was born prematurely with a family history typical of HPS. Urinary salt and water losses and PGE(2) excretion were determined in 2- to 4-h intervals. Salt and water were replaced accordingly. RESULTS: Within the first 48 h, urinary losses and PGE(2) increased continuously to 50 ml/kg/h and 374 ng/h/1.73 m(2), respectively. Following exposure to 0.05-0.5 mg/kg/d indomethacin, urinary output decreased steadily to 10-15/ml/kg/h. CONCLUSION: In euvolaemic preterm neonates with HPS and the need for excessive replacement of salt and water, inhibition of renal PGE(2) excretion with indomethacin effectively reduces polyuria and natriuresis without acutely compromising renal function.

Expression of the potassium channel ROMK in adult and fetal human kidney.

The renal potassium channel ROMK is a crucial element of K+ recycling and secretion in the distal tubule and the collecting duct system. Mutations in the ROMK gene (KCNJ1) lead to hyperprostaglandin E syndrome/antenatal Bartter syndrome, a life-threatening hypokalemic disorder of the newborn. The localization of ROMK channel protein, however, remains unknown in humans. We generated an affinity-purified specific polyclonal anti-ROMK antibody raised against a C-terminal peptide of human ROMK. Immunoblotting revealed a 45 kDa protein band in both rat and human kidney tissue. In human kidney sections, the antibody showed intense staining of epithelial cells in the cortical and medullary thick ascending limb (TAL), the connecting tubule, and the collecting duct. Moreover, a strong expression of ROMK protein was detected in cells of the macula densa. In epithelial cells of the TAL expression of ROMK protein was mainly restricted to the apical membrane. In human fetal kidney expression of ROMK protein was detected mainly in distal tubules of mature nephrons but not or only marginally in the collecting system. No expression was found in early developmental stages such as comma or S shapes, indicating a differentiation-dependent expression of ROMK protein. In summary, these findings support the proposed role of ROMK channels in potassium recycling and in the regulation of K+ secretion and present a rationale for the phenotype observed in patients with ROMK deficiency.

Salt handling in the distal nephron: lessons learned from inherited human disorders.

The molecular basis of inherited salt-losing tubular disorders with secondary hypokalemia has become much clearer in the past two decades. Two distinct segments along the nephron turned out to be affected, the thick ascending limb of Henle's loop and the distal convoluted tubule, accounting for two major clinical phenotypes, hyperprostaglandin E syndrome and Bartter-Gitelman syndrome. To date, inactivating mutations have been detected in six different genes encoding for proteins involved in renal transepithelial salt transport. Careful examination of genetically defined patients ("human knockouts") allowed us to determine the individual role of a specific protein and its contribution to the overall process of renal salt reabsorption. The recent generation of several genetically engineered mouse models that are deficient in orthologous genes further enabled us to compare the human phenotype with the animal models, revealing some unexpected interspecies differences. As the first line treatment in hyperprostaglandin E syndrome includes cyclooxygenase inhibitors, we propose some hypotheses about the mysterious role of PGE(2) in the etiology of renal salt-losing disorders.

Dominant role of prostaglandin E2 EP4 receptor in furosemide-induced salt-losing tubulopathy: a model for hyperprostaglandin E syndrome/antenatal Bartter syndrome.

Increased formation of prostaglandin E2 (PGE2) is a key part of hyperprostaglandin E syndrome/antenatal Bartter syndrome (HPS/aBS), a renal disease characterized by NaCl wasting, water loss, and hyperreninism. Inhibition of PGE2 formation by cyclo-oxygenase inhibitors significantly lowers patient mortality and morbidity. However, the pathogenic role of PGE2 in HPS/aBS awaits clarification. Chronic blockade of the Na-K-2Cl co-transporter NKCC2 by diuretics causes symptoms similar to HPS/aBS and provides a useful animal model. In wild-type (WT) mice and in mice lacking distinct PGE2 receptors (EP1-/-, EP2-/-, EP3-/-, and EP4-/-), the effect of chronic furosemide administration (7 d) on urine output, sodium and potassium excretion, and renin secretion was determined. Furthermore, furosemide-induced diuresis and renin activity were analyzed in mice with defective PGI2 receptors (IP-/-). In all animals studied, furosemide stimulated a rise in diuresis and electrolyte excretion. However, this effect was blunted in EP1-/-, EP3-/-, and EP4-/- mice. Compared with WT mice, no difference was observed in EP2-/- and IP-/- mice. The furosemide-induced increase in plasma renin concentration was significantly decreased in EP4-/- mice and to a lesser degree also in IP-/- mice. Pharmacologic inhibition of EP4 receptors in furosemide-treated WT mice with the specific antagonist ONO-AE3-208 mimicked the changes in renin mRNA expression, plasma renin concentration, diuresis, and sodium excretion seen in EP4-/- mice. The GFR in EP4-/- mice was not changed compared with that in WT mice, which indicated that blunted diuresis and salt loss seen in EP4-/- mice were not a consequence of lower GFR. In summary, these findings demonstrate that the EP4 receptor mediates PGE2-induced renin secretion and that EP1, EP3, and EP4 receptors all contribute to enhanced PGE2-mediated salt and water excretion in the HPS/aBS model.