Role of Microsomal Prostaglandin E Synthase 1 in the Kidney Helene Francois,* Carie Facemire,* Anil Kumar,* Laurent Audoly,† Beverly Koller,‡ and Thomas Coffman* *Divisions of Nephrology, Duke University and Durham Veterans Affairs Medical Centers, Durham, North Carolina, †MedImmune, Gaithersburg, Maryland, and ‡Department of Genetics, University of North Carolina, Chapel Hill, North Carolina Prostaglandin E2 (PGE2) is one of the most ubiquitous prostanoids in the kidney, where it may influence a wide range of physiologic functions. PGE2 is generated through enzymatic metabolism of prostanoid endoperoxides by specific PGE synthases (PGES). Several putative PGES have been identified and cloned, including the membrane-associated, inducible microsomal PGES1 (mPGES1), which is expressed in the kidney. To evaluate the physiologic role of mPGES1 in the kidney, mice with targeted disruption of mPges1 gene were studied, with a focus on responses where PGE2 has been implicated, including urinary concentration, regulation of blood pressure, and response to a loop diuretic. The absence of mPGES1 was associated with a 50% decrease in basal excretion of PGE2 in urine (P < 0.001). In female but not male mPGES1-deficient mice, there was a reciprocal increase in basal excretion of other prostanoids. Nonetheless, urinary osmolalities were similar in mPges1؉/؉ and mPges1؊/؊ mice at baseline and after 12 h of water deprivation. Likewise, there were no differences in blood pressure between mPGES1-deficient and wild-type mice on control or high- or low-salt diets. The furosemide-induced increase in urinary PGE2 excretion that was seen in wild-type mice was attenuated in mPGES1-deficient mice. However, furosemide-associated diuresis was reduced only in male, not female, mPGES1-deficient mice. Stimulation of renin by furosemide was not affected by mPGES1 deficiency. These data suggest that mPGES1 contributes to basal synthesis of PGE2, but there are other pathways that lead to renal PGE2 synthesis. Moreover, there are significant gender differences in physiologic contributions of mPGES1 to control kidney function. J Am Soc Nephrol 18: 1466–1475, 2007. doi: 10.1681/ASN.2006040343 rostaglandin E2 (PGE2) is one of the major prostanoid the actions of phospholipases, conversion of arachidonic acid to products generated by the kidney (1), and it has diverse unstable endoperoxide intermediates by cyclooxygenases actions in the kidney, affecting both vascular tone and (COX-1 and -2), and, finally, isomerization of PGH to PGE by P 2 2 epithelial functions. For example, infusion of PGE2 into the terminal PGE synthases (PGES) (8). PGES activity has been kidney commonly causes renal vasodilation (2,3). PGE2 also characterized biochemically in a number of tissues (9). In most modulates renal sodium and water excretion (1,4). Studies in cell populations, the highest levels of specific PGES activity are isolated perfused nephron segments suggest that PGE2 acting associated with microsomal membrane fractions. Jakobsson through E-prostanoid (EP) receptors such as EP1 may directly and associates (10,11) provided the first molecular character- stimulate solute flux in renal epithelia (5). PGE2 may also ization of a membrane-bound form of human PGES (mPGES1), influence water permeability in the distal nephron by opposing showing that it is a member of the MAPEG (membrane-asso- the actions of the stimulated G-protein–linked vasopressin re- ciated proteins involved in eicosanoid and glutathione metab- ceptor (4). Moreover, enhanced generation of PGE2 seems to olism) gene family. Expression of mPGES1 is highly inducible contribute to the actions of loop diuretics (6). It has been sug- in states of injury and inflammation, often in parallel with gested that the propensity of nonsteroidal anti-inflammatory expression of the inducible COX-2 (12,13). More recently, a drugs (NSAID) to cause fluid retention and hypertension is due second putative microsomal PGES (mPGES2) that can also to inhibition of PGE synthesis and consequent attenuation of 2 generate PGE from endoperoxides in vitro with high affinity its actions in the kidney (7). 2 and specificity was identified (14,15), but its capacity to gener- PGE is produced by a series of three enzymatic reactions: 2 ate PGE in vivo is not clear. A third form of PGES has been release of arachidonic acid from membrane phospholipids by 2 isolated from cytoplasmic extracts (16). This cytosolic PGES (cPGES) is expressed ubiquitously in a constitutive manner Received April 11, 2006. Accepted March 30, 2007. (16). However, because of its low substrate affinity compared with the microsomal enzymes, the relevance of cPGES to the Published online ahead of print. Publication date available at www.jasn.org. generation of PGE2 in vivo has also been questioned. Address correspondence to: Dr. Thomas M. Coffman, Building 6/Nephrology (111I), VA Medical Center, 508 Fulton Street, Durham, NC 27705. Phone: 919-286- Very little is known about the functions of the individual 6947; Fax: 919-286-6879; E-mail: [email protected] PGES in the kidney. Nonetheless, several studies have evalu- Copyright © 2007 by the American Society of Nephrology ISSN: 1046-6673/1805-1466 J Am Soc Nephrol 18: 1466–1475, 2007 mPGES1 and Kidney Function 1467 ated regional expression of mPGES1 in the kidney by in situ in phosphate and carbonate buffer prepared with deionized water and hybridization and immunocytochemistry (5,17–19). There is a incubated overnight at 37°C to convert intact PGE2 and its intermediate general agreement among these studies that mPGES1 is ex- metabolites to the stable PGE2 metabolite (PGEM). Concentrations of pressed in the distal nephron from the connecting tubule PGEM in urine were determined by using a specific ELISA assay (Cayman Chemicals, Ann Arbor, MI). Previous studies have shown through the cortical and medullary collecting duct (5,17–19). In that urinary PGEM is a reliable indicator of urinary PGE excretion in this region, the distribution of mPGES1 parallels COX-1 expres- 2 vivo (24). Thromboxane B (TxB ), the stable metabolite of TxA , and sion (18,19). This distribution is consistent with previous stud- 2 2 2 6-keto- PGF1␣, the stable metabolite of prostacyclin (PGI2), were mea- ies indicating that the collecting duct is one of the nephron sured in fresh urine using specific ELISA (Cayman Chemicals). segments with the highest capacity for PGE2 generation (1); abundant EP receptors, including EP1, EP3, and EP4, are also Assessment of Urinary Concentrating Capacity expressed in the collecting duct (4,20). Moreover, this segment Mice were placed in metabolic cages for collection of urine and of the nephron plays a critical role in the final adjustments of monitoring of water intake. After an initial adaptation period, water sodium excretion that have a major impact on fluid volume and intake and urine volumes were measured while the mice had free ϩ ϩ Ϫ Ϫ BP homeostasis. However, the capacity of individual mPGES to access to water. Urine of male and female mPges1 / and mPges1 / influence renal functions is not known. mice was collected by bladder massage before and after a 12-h period In studies that used mPGES1-deficient mice, we show here of water deprivation. Urine osmolalities were then immediately mea- that mPGES1 contributes to basal and stimulated PGE2 gener- sured using a vapor pressure osmometer (Wescor Instruments, Logan, ation by the kidney. In male mice, augmented generation of UT) as described previously (25). PGE2 by mPGES1 contributes to furosemide-induced diuresis. However, the relative contribution of mPGES1 to synthesis and Responses to the Loop Diuretic Furosemide functional consequences of PGE2 in the kidney differs signifi- A 5-mg/ml furosemide (Sigma Chemicals, St. Louis, MO) solution cantly between genders. was prepared in 10% DMSO in deionized water at pH 7. The 10% DMSO solution alone was used for control injections. Mice were ad- Materials and Methods ministered an injection of 0.1 ml/10 g body wt of the furosemide Animals solution or vehicle control and were immediately placed into individ- Production of mice with targeted disruption of the mPges1 gene has ual metabolic cages, where they were provided free access to tap water. been described previously (21). The mPges1 mutation was originally Urine was collected for exactly 4 h. Urine volumes were measured generated by homologous recombination in embryonic stem (ES) cells using tared containers, and urine sodium concentrations were mea- that were derived from DBA/1 inbred mice. Resulting chimeras were sured using a flame photometer (Instrumentation Laboratory, Lexing- crossed with DBA/1 mice (Jackson Laboratory, Bar Harbor, ME) to ton, MA). For definition of the contribution of prostanoids to the generate inbred DBA/1 mice that bear the mPges1 mutation. Inbred furosemide response, separate groups of inbred male and female mice Ϫ Ϫ ϩ ϩ DBA/1 mPges1 / and mPges1 / mice that were generated by inter- were given diclofenac (10 mg/kg; Sigma Chemicals) in drinking water ϩ Ϫ crossing DBA/1 mPges1 / mice were used for the studies described for 24 h before the furosemide injection. here. Mice were bred and maintained in the animal facility of the Durham Veterans Affairs Medical Center, and genotypes were deter- Reverse Transcription and Real-Time Quantitative PCR mined as described previously (21). The experimental procedures de- Total RNA was extracted from whole kidneys using TriReagent scribed next were approved by the respective institutional animal care (Sigma) according to the manufacturer’s protocol. RNA was DNAse- and use committees of the Durham Veterans Affairs and Duke Univer- treated using Turbo DNA-free (Ambion, Austin, TX) to remove genomic sity Medical Centers. DNA contamination. RNA yield was quantified by ultraviolet spectro- photometry, and integrity was verified by 1% agarose gel electrophore- Measurement of Systolic BP in Mice sis and staining with ethidium bromide. Only RNA with A260/280Ͼ1.7 Systolic BP was measured in conscious mice using a computerized and displaying no significant degradation was used for reverse tran- tail-cuff system (Hatteras Instruments, Cary, NC) after 2 wk of daily scription.