Metabolism of Prostaglandins in the Kidney

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Kidney International, Vol. 19 (1981), pp. 760—770

Metabolism of prostaglandins in the kidney

FRANK F. SUN, BRUCE M. TAYLOR, JAMES C. MCGUIRE, and PATRICK Y. K. WONG

Department of Experimental Biology, The Upjohn Company, Kalamazoo, Michigan, and Department of Pharmacology, New York Medical College, Valhalla, New York

Since the early report of Lee et a! [1] describing a recommend that the reader consult the reviews vasoactive acidic lipid in the rabbit renal medulla, published elsewhere [4—6]. the kidney has been recognized as a major site of prostaglandin metabolism in the body. In fact, the Prostaglandin biosynthesis kidney medulla is one of the richest sources of The renal prostaglandin biosynthesis has been cyclooxygenase activity and, therefore, has been extensively investigated for over a decade [7—9], but widely used for prostaglandin biosynthetic studies. there are still many gray areas of ambiguity. In Many direct and indirect findings have established vitro, the renal papilla and medulla from rat, rabbit, that PG!2 and PGE2 play important roles in the and human [7-46] contain more cyclooxygenase regulation of renal blood flow and maintenance of (prostaglandin endoperoxide synthetase) than the electrolyte balance. Other oxygenated metabolites cortex does. The medullary cyclooxygenase is of arachidonic acid (that is, PGF2a, PGD2, TXB2, membrane bound and can be solubilized and isolat- hydroxyeicosatetraenoic acids, and leucotrienes ed by column chromatography [14]. The solubilized [2]) are reportedly synthesized by the kidney, al- enzyme has a heme-dependent cyclooxygenase ac- though their function remains as yet undefined. tivity that converts arachidonic acid to the hydro- Tissues do not store prostaglandins but generate peroxide PGG2 and a peroxidase activity that con- them from endogenous substrate fatty acids on verts PGG2 to PGH2. The overall process is stimu- demand. Several peptide hormones including bra- lated by phenolic compounds, but is inhibited by dykinin and angiotensin II are able to activate renal nonsteroidal antiinflammatory agents. In vivo, the phospholipase and release arachidonic acid for activity is probably limited by substrate availability prostaglandin synthesis. The action of prostaglan- [17—2 1]. Experiments with cx vivo isolated perfused dins is usually limited to their site of biosynthesis rabbit kidneys [19—21] have shown that bradykinin because powerful prostaglandin dehydrogenases and angiotensin II, which stimulated phospholipase are present, particularly in the lung, that inactivate activity, lead to increased PGE2 release. Similar the PG's before their removal from circulation. experiments with rat papilla slices [17] or isolated The kidney is also an important organ for prosta- renomedullary interstitial cells [18] in vitro or dog glandin excretion. It accumulates exogenous pros- kidney in vivo [221 have yielded similar findings. taglandins above the plasma level [3] and is respon- Anatomically, the cyclooxygenase-containing sible for removing 50 to 80% of the body's prosta- cells in the kidney of several species have been glandins by the urinary route, although very little is located by an immunofluorescence procedure [23— yet known about specific mechanisms. This article 25].In all species, the enzyme was found in the will present a brief review of the recent information epithelial cell of the cortical and medullary collect- on the biosynthesis and metabolism of renal prosta- ing ducts, arterial vascular endothelial cells, and glandins. For a more extensive discussion, we medullary interstitial cells. Other sites containing cyclooxygenase include the parietal layer of Bow- man's capsule in rabbit, mesangial cells in ovine and bovine glomeruli, and the thin limbs of Henle's Received for publication December 11, 1980 ioop in the hydronephrotic rabbit kidney. Some of 0085-2538/81/0019-0760 $02.20 these cells or structures have been isolated and ©1981 by the International Society of Nephrology their PG synthetases studied by biochemical meth-

760 Renal metabolism of prostaglandins 761 ods. In this respect, interstitial cells from rat and strate affinity [39] and cofactor requirements [40]. rabbit [18, 26, 27] have been reported to produce Therefore, any difference in substrate concentra- PGE2 and PGF2,. Terragno, McGiff, and Terragno tion, incubation time, or the presence of cofactors [281 demonstrated that the arteries and arterioles used in each study may lead to differences in isolated from pig kidney cortex by microdissection prostaglandin product distribution. Furthermore, have a high capacity for PG!2 biosynthesis. Bohman prostaglandin endoperoxides are extremely unsta- [29] and Grenier and Smith [30] have reported that ble. If the cyclooxygenase in an enriched tissue the collecting duct epithelial cells isolated from such as kidney medulla is allowed to operate at rabbit renal papilla synthesized 6-keto-PGF1, optimal condition, the endoperoxide generated may PGF2a, PGE2, and PGD2. Other groups [3 1—33] be more than its isomerases can consume. The have shown that the isolated glomeruli from rat excess will randomly decompose to PGF2a, PGE2, kidney cortex have the capacity for synthesis of and PGD2. Our in vitro study described below best PGF2a, PGE2, 6-keto-PGF1a, thromboxane B2, and illustrates this situation. PGD2. The mesangial cells [34] and the epithelial When a piece of rabbit inner medulla was homog- cells [351 are the site of prostaglandin E2 biosynthe- enized with '4C-arachidonate and incubated for 5 sis, and the arterioles probably produce PGI2, mm, the formation of radioactive prostaglandin whereas the cortical tubule fragments [311 contain products varied with the substrate concentration much lower activity than do the glomeruli. (Fig. 1). If the exogenous arachidonic acid was The literature shows qualitative and quantitative sufficiently low (2 pM) the product was almost inconsistencies in prostaglandins produced in dif- exclusively PGE2. If larger amounts (0.1 #.LM) of ferent regions of the kidney. Whorton et al [15] substrate were added or the cyclooxygenase was demonstrated clear evidence that rabbit kidney stimulated by phenolic compounds, the products cortex microsomes synthesized both PGI2 and were PGE2, PGF2a, and PGD2. Addition of reduced PGE2 from arachidonic acid and PG endoperoxide, glutathione increased PGE2 at the expense of PGF2a whereas the medullary microsomes synthesized ex- and PGD2. These results suggest that the enzymatic clusively PGE2. Similar experiments with rat inner product in the medulla is PGE2, and the rate- medullary slices [36] or rabbit medulla microsomes limiting step is the PGE2 isomerase. When the [14] agreed with these results. New evidences, cyclooxygenase is artificially stimulated, any ex- however, extended these observations and showed cess endoperoxide decomposes into a variety of that the medulla also had the capacity of synthesiz- mixed prostaglandin products. But if the isomerase ing PG!2. Silberbauer and Sinzinger [37] found that is stabilized with reduced glutathione, the enzyme both cortex and medulla of rat generated prostacy- can handle additional amounts of endoperoxide. din-like activity blocking platelet aggregation and Throughout this study, the formation of 6-keto- that the medulla was significantly more active than PGF1, was not detected. the cortex. With a gas chromatography mass spec- A small amount of 1 l-hydroxyeicosatetraenoic trometry method, Oliw et al [38] found high levels acid (1 1-HETE) was detected in the extract and its of 6-keto-PGF1a accumulated in cortex, medulla, identity was confirmed by GCMS analysis. This and papilla of postmortem rabbit kidney. Although confirmed a similar finding of Hamberg [7]. The 11- the papilla 6-keto-PGF1a level was the highest, the HETE was not a product of lipoxygenase because cortex contained five times more 6-keto-PGF1a than its formation can be inhibited by indomethacin. It is did PGE2 and PGF2a. Very recently, Hassid and probably a side product of the cyclooxygenase as Dunn [16] reported that microsomes prepared from suggested by Porter et al [411. both cortex and medulla of human kidney convert- Similar experiments with cortex slices yielded ed arachidonic acid to PGF2a, 6-keto-PGF1a, TXB2, smaller amounts of prostaglandins. With either low and PGE2 and that the relative abundances were or high substrate concentration, the cortex pro- similar in both regions. They concluded that there duced four times less prostaglandins than did the was no compartmentalization of PG!2 synthetase in medulla. PGE2 and PGF2a were the main products, the cortex. and very little 6-keto-PGF1a was detected. We feel these inconsistencies arose from differ- Although these results agreed with earlier report- ences in the in vitro experimental conditions. The ed work, we suspected that the PG!2 synthetase in medulla cortex and papilla probably contain isomer- our preparation was being inactivated by the organ- ases for the production of PGE2 and PG!2. The two ic peroxides generated during vigorous tissue ho- enzymes, however, have wider differences in sub- mogenation in air. Therefore, we prepared washed 762 Sunet a!

Medulla Medulla 2 nmole AA 1000 nmole AA

6KF 6KF

Cortex b Medulla d 2 nmole AA 1000 nmole AA + glutathione

6KF 6KF

Fig. 1. Radiochromatogram of the extract from the conversion of'C-arachidonic acid to prostaglandins by rabbit kidney homogenate: a medulla 1.0 g of tissue and 2 LMAA,b cortex 1.0 g of tissue and 2 I.LM AA, cmedulla0.1 g of tissue and 100 IJ.M AA, and d medulla 0.1 g of tissue, 100 ILM AA, and I m reduced glutathione. microsomes from both rabbit cortex and medulla suggesting that the Km values for the two enzymes under an argon atmosphere. Indomethacin was also are different. At all substrate concentrations, the added to stop PG biosynthesis. These microsomes yield of 6-keto-PGF1a from medullary microsome were incubated with various concentrations of '4C- was three to four times greater than the cortex PGH2, and the products were analyzed by thin- microsome was. layer chromatography (Fig. 2). Therefore, it is clear that the PGI2 synthetase in At the lowest PGH2 concentration (0.4 pM),the the kidney is sensitive to harsh tissue handling. medulla and cortex microsomes both produced Unprotected homogenization and cell fractionation abundant amounts of 6-keto-PGF1. The yield of probably activate cyclooxygenase and generate or- PGF2a, PGE2, and PGD2 were identical to the heat- ganic peroxides that inactivate PGI2 synthetase. If inactivated enzyme control. Addition of reduced properly protected, the in vitro measurement of glutathione to the medulla microsome reaction sig- PGI2 biosynthesis in microsomes corresponds very nificantly increased the yield of PGE2, whereas in well with the results from tissue slices [37] or the cortex case it increased PGF2a. Figure 3 illus- postmortem accumulation of PGI2 in vivo [381. It trates the effect of increasing the PGH2 concentra- should be noted that the difference in PGI2 synthe- tion on the activities of cortical and medullary PGI2 tase activities between cortex and medulla parallels synthetase and the glutathione-dependent medul- closely their cyclooxygenase activities. lary PGE2 isomerase. Both prostacyclin synthetase To assess the classical prostaglandins formed reactions follow a hyperbolic pattern and approach enzymatically from PGH2 requires a precise com- saturation as PGH2 concentration reaches 10 .LM. parison between active microsome and its heat- Medullary PGE2 synthesis continued to increase inactivated control. The amounts of prostaglandin with increasing PGH2 concentration beyond 15 .LM, E2, D2, and F2 formed after incubation of PGH2 Renal metabolism of prostaglandins 763

Medu la Cortex

b d

Medulla Cortex + glutathione + glutathione

6KF

Fig.2. Radiochromatogram of the extract from the conversion of '4C-PGH2 (0.44 M) to prostagiandins by rabbit kidney microsome prepared in the presence of 0.1 mi indomethacin and under the atmosphere of argon: a medulla 1 mg/kg, b medulla I mg/mi, 1 mM reduced glutathione, c cortex 4 mg/mi, and d cortex 4 mg/mi, 1 mrei reduced glutathione. with heat-inactivated enzyme were substracted appear to be quite similar to the PGE2 isomerase from the active microsome products. After this isolated by Ogino et al [401 from bovine vesicular correction, only the glutathione-dependent PGE2 gland. synthesis in the medulla and glutathione-dependent The data clearly indicate that PGE2 and PGI2 are PGF2a synthesis in the cortex appeared to be cata- the principal cyclooxygenase products in normal lyzed by enzymes. We have developed a simple rabbit kidney. The formation of PGD2 and a great assay to measure the medullary PGE isomerase portion of PGF2a were decomposition products of activity. Labeled PGH2 was incubated with medulla excess endoperoxide in an in vitro heterogenous microsome for 30 seconds, and the reaction was surrounding. The distribution of PGI2 synthetase quenched by the addition of ferrous chloride to parallels the distribution of cyclooxygenase in vari- convert unreacted PGH2 to HHT. The yield of ous regions of the kidney. It is possible that the PGE2 is measured by radio thin-layer chromatogra- endoperoxide metabolizing enzymes are coupled phy. In this assay, the reduced glutathione is an with specific cyclooxygenase for efficient and rapid essential cofactor. The requirement can be replaced conversion of the released arachidonic acid to a by the high-speed supernatant from renal medulla, specific product [211. indicating the presence of endogenous cofactors. But, the requirement cannot be fulfilled by cys- Prostaglandin catabolism teine. In contrast to the PGI2 synthetase, the PGE2 The kidney possesses enzymatic activities for isomerase is not inhibited by azo analog I (U- essentially every major step of prostaglandin catab- 51,605). The properties of the renal PGE2 isomerase olism. These reactions include C-iS oxidation, 13 764 Sun et al

oxidation of the 15(S) hydroxyl group of the E, F, A, and I series of prostaglandins to their 15-keto derivative. PGB, PGD, 6-keto-PGF1 or thromboxane B2 are poor substrates. In contrast to the type II PGDH, the type 1 enzyme has a very rigid substrate specificity and the reaction is essentially irreversible. The tubular location of the renal PGDH plus the fact that the enzyme does not act on circulating prostaglandins (see later sections) suggests that the enzyme may play a role as a regulator of intrarenal prostaglandin activities. In this connection, it is interesting to note that the diuretic action of furose- mide and ethacrylic acid has been related to their inhibition of renal PGDH [45, 46]. In the hypertensive rat, the activity of renal PGDH was significantly lower than that of the normotensive control [48]. This finding has been related to the increased vascular resistance in the hypertensive animals. Renal PGDH also has a short life span, and its replacement depends on de novo protein synthesis [491. Presumably its activity could be controlled by blood-born factors that affect protein synthesis. (2) Type ii prostaglandin dehydrogenase. Type 5.0 10.0 15.0 II PGDH is a relatively nonspecific enzyme catalyz- PGH2added, X 10 M ing the reversible interconversion of hydroxyl and Fig. 3. Effect of substrate concentrations on the formation of keto functions at both C-9 and C-iS. Previously PG!2 and PGE2 (glutathione dependent) by rabbit renal medullary microsome and PG!2 by rabbit renal cortical rnicrosorne—l described as two separate enzymes, 9-keto reduc- ml of microsome (0.8 mg/rn/for medulla and 4 mg/rn/for cortex) tase and NADP-dependent 15-hydroxy prosta- with various concentrations of'4C-PGH2 for! mm at 370 C. The glandin dehydrogenase, it now appears that the two products were extracted and analyzed by thin layer chromatography (organic layer of EtoAC:HAc: 2,2,4-trimethyl pen- activities are associated with a single protein spe- tane:H,0, 110:20:50:100). cies. The enzyme has been purified from the kidneys of swine [50,51],chicken [46], rabbit [52], and monkey (Sun and Cray, unpublished findings), as reduction, p-oxidation, o-hydroxylation, and inter- well as other organs or tissues from a number of conversion of hydroxyl and keto groups at C-9. species. In most cases, the enzyme existed in Most of the knowledge was obtained by in vitro several molecular forms that can be resolved by experimentation with tissue homogenate. Little in- isoelectric focusing. The cofactor requirement is formation exists on the roles of kidney as a metabol- NADP, and the enzyme is fully reversible at both ic organ in vivo. the C-9 and the C-IS position. The substrate speci- Four types of prostaglandin oxidoreductases ficity of the partially purified monkey kidney type II have been found in the high-speed supernatant of PGDH is summarized in Table 1. Despite the appar- kidney homogenate. Their properties are summa- ent broad specificity, the enzyme will not reduce rized in the following sections. the 1 1-keto of PGD2. In a recent study of Chang and (1) Type I 15-hydroxy prostaglandin dehydrogen- Tai [50], a purified swine kidney type II PGDH ase (PGDH). The type I PGDH catalyzes the first preparation failed to reduce 6-keto-PGF1. The step of prostaglandin inactivation. It occurs most higher activities of 15-methyl PGE2 and 16,16- abundantly in the cortex [42], and histochemical dimethyl PGE2 suggested that the reaction centers staining has located the enzyme in the proximal and at C-9 and C-IS might compete with each other for distal tubules [43, 441.RenalPGDH from rat [45], the same active site on the enzyme surface. Elimi— chicken [46], pig [47], and monkey have been nation of the C-IS reaction facilitates the reaction of purified, and multiple species have been detected C-9. The specificity apparently is not confined to for the chicken enzyme. The enzyme catalyzes the prostaglandin moieties because the enzyme also Renal metabolism of prostaglandins 765

Table 1. Substrate specificity of purified type II PGDH from The enzyme has been purified from bovine lung [57] monkey kidney and human placenta [58] but the renal enzyme has Relative activity not been studied in detail. Reduction NADPH as cofactor (4) 9-Hydroxy prostaglandin dehydrogenase. PGE2 100 This is a unique enzyme different from the type II PGE1 62 to 70 PGDH. It was found in the cortex of kidneys of rat PGD1 0 [59] and rabbit [60]. The enzyme catalyzes the PGD2 0 PGF2, 0 oxidation of 9ct-hydroxyl groups of prostaglandin PGA2 48 to 59 F2a and Fia, but not 6-keto-PGF1c. or PGF2. The PGB2 24 to 34 significance of this conversion is unclear. 15-Keto dihydro PGF2. 56 to 90 15-Keto PGF2 20 to 68 It should be mentioned that a separate 9-hydroxyl 15-Methyl PGE2 143 to 244 PGDH [61] was detected in rabbit liver and human 16,16-Dimethyl PGE2 143 to 230 platelet. The enzyme oxidizes 6-keto-PGF1a to the Sct-Dihydro testosterone 150 to 400 Oxidation NADP as cofactor biologically active molecule 6-keto-PGE1. The kid- PGF2 332 ney apparently contains little of this enzyme. PGE2 100 The presence of fatty acid w and w-1 hydroxyl- PGE1 160 16, 16-Dimethyl PGE2 0 ation system in kidney cortex microsome of rat and 15-Methyl PGE2 0 pig has been demonstrated [62], although little work has been reported. The n-oxidation of fatty acid is a universal process occurring in all major organs including the kidney. catalyzedthe rapid reduction of 5a-dihydrotestos- terone in the presence of NADPH, arid the steroid In vitro studies reductase activity cannot be separated from the PG A number of studies have been conducted to reductase activity throughout the purification. The examine the prostaglandin metabolism in crude results suggest that the same enzyme may be re- kidney homogenate. In most in vitro studies, the sponsible for the oxidation-reduction of both types reaction mixture is fortified with pyridine nucleo- of molecules. tides [63—65]. The direction of the metabolic se- The type II PGDH occurs in both cortex and quence was thus determined by the cofactor added. medulla [51] of the swine kidney. It has a much Therefore, incubation of labeled PGE2, PGF2. or lower affinity for prostaglandins than the type I PGI2 with kidney supernatant of rat and rabbit in enzyme does. Apparent Kms for reduction of 9- the presence of NAD gave predominantly their keto group of PGE2 are 400 LM for enzyme from 13,14-dihydro 15-keto metabolites. But, if NADPH both rabbit [521 and monkey (Sun and Cray, unpub- were added, PGE2 would be reduced to PGF2. This lished findings). The swine kidney type I PGDH indicates that the direction of PG catabolism in vivo binds to the PG-attached sepharose whereas type II is a function of intracellular pyridine nucleotide does not [53]. But, the reactions catalyzed by the ratio and their redox state. Moreover, species dif- type II PGDH were observed in in vivo prostaglan- ferences exist in the levels and activities of various din metabolism [54, 551.Increased9-keto reductase cytoplasmic enzymes. Hoult and Moore [64] stud- levels were reported in the kidney of chronically ied the oxidative metabolism of PGF2a in kidney salt-loaded rabbits [561. The idea remains viable supernatants of rat, rabbit, guinea pig, and pig and that the enzyme may be another site for regulating found the metabolites were both qualitatively and prostaglandin activity. quantitatively different. The major product was (3) 15-Keto prostaglandin z'3-reductase. In as- 13,14-dihydro-15-keto PGF2a, but the rat also oxi- sociation with the type I PGDH, the 13reductase dized the dihydro keto PGF2 to dihydro keto PGE2 reduces the &doublebond of the l5-keto prosta- whereas guinea pig and pig reduced it to dihydro glandins to the 13,14-dihydro 15-keto prostaglan- PGF2a. Only in the rabbit were substantial amounts dins. The cofactor can be either NADH or of PGF2a oxdized directly to PGE2. The metabolic NADPH, and the reaction is irreversible. The en- reaction sequence is summarized in Fig. 4. zyme is specific for the 15-keto prostaglandins of the E, F, D, and I series and probably the 15-keto Ex vivo studies thromboxane B2. It does not catalyze the reduction Despite the extensive studies of prostaglandin of their corresponding 15-hydroxyl prostaglandins. metabolism in vitro, little information is published 766 Sun et a!

OH OH

H H N AD* a',-

OH OH OH° I*iiuuuuuuiiiiiiuuiic00

NADP

OHO

NADPH

Fig. 4. Invitro prostaglandin (PG) metabolism by rabbit kidney supernatant. Thedirection of PG metabolism may be a function of intracellular pyridine nucleotide ratio and their redox state. on the fate of renal prostaglandins in vivo, particu- pass perfusion. The venous effluent and in some larly the events involved in the renal handling of cases urine samples were collected separately and circulating prostaglandins. PGE2 and PGF2a inject- analyzed. ed into the renal artery of the isolated perfused When multiple tritium labeled PGE2 was infused rabbit kidney are filtered through the glomerulus into the perfused kidney, 80 to 85% of the injected and secreted by the tubules 166—681. The latter radioactivity was recovered in the perfusate. The process is mediated by the organic anion transport perfusate was passed through an Amberlite XAD-2 system and can be blocked by indomethacin, pro- column. Seventy-five to eighty percent of radioac- benecid, or bromocresol green. Significant metabo- tivity was extractable as lipid material, and the lism does occur during the tubular transport. Pros- remaining radioactivity was water soluble material taglandin E2 is also absorbed by the loop [69] of or tritiated water. The metabolites were separated Henle and the distal tube in the rat. The intact by high-performance liquid chromatography and prostaglandins found in the urine are believed to be analyzed by gas chromatography-mass spectrome- of renal origin [70], with the point of entry most try. In the perfusate, unmetabolized PGE2 was 16 to likely Henle's loop [711. 31% whereas the major metabolite was the tetranor We have investigated the prostaglandin renal PGF (Fig. 5). The formation of the tetranor PGF metabolism in an ex vivo perfused rabbit kidney from PGE2 involves f3-oxidation and reduction of preparation. The method of Schurek et al [721 was the keto group at C-9. A small amount of tetranor modified for rabbit kidneys, and the prostaglandins 15-keto dihydro PGF was also found, which indicat- were delivered at a rate of 10 p.g/min during a single- ed that the 15-PGDH and reductase routes were Renal metabolism of prostaglandins 767

0 "COOH 69-84% + conversion - -

OH OH Trace >90% PGE2

PG F20 >90% —1.2% HOOC

OH OH

OH OH 60% OH OH OH OH 30%

PG 12

Fig. 5. Proposed metabolic pathways of PGE2, PGF2, and PG!2 in isolated perfused rabbit kidney. The stereo configuration of C-9 hydroxyl from reduction of PGE2 has not been determined. of minor importance. The metabolic profile was only a minor product in the vascular perfusate; unchanged by increasing the cytosolic pyridine nu- sometimes less than 5% of the total perfu sate radio- cleotide oxidation potential by adding pyruvate and activity. red blood cells [73] to the perfusion fluid. There- Because it is unlikely that large amounts of intact fore, the extensive reduction of the 9-keto group prostaglandins would be delivered to the kidney for probably was not a result of anoxia-induced reduc- metabolism, we also examined the kidney handling tion of pyridine nucleotide. of major circulating prostaglandin metabolites. Ra- Extensive 13-oxidation was also observed for diolabeled 13,14-dihydro-l5-keto PGE7 and 13,14- PGF2a and PGI2. Consistent with the PGE2 study, dihydro- I 5-keto PGF20 were infused into the rabbit the 15-PGDH products in venous effluent were kidney and the metabolic products analyzed by gas scarce or absent. PGF2c. was almost completely chromatography mass spectrometry (Fig. 6). For (97%) converted to tetranor PGF10 during a single these compounds, the overall metabolic conversion pass. No PGE metabolites were detected, indicat- (63 to 64%) was less than their parent compounds. ing that the 9-hydroxydehydrogenase was not oper- Perhaps the metabolites were not efficiently trans- ating. PGI2 was more resistant to 13-oxidation, form- ported from vascular compartment into the cells. ing only 30 to 50% dinor metabolites. Apparently The metabolites from 15-keto-dihydro-PGE2 were the cyclic enol ether or the 6-keto functional groups tetranor dihydro PGE1, tetranor dihydro PGF, and retarded 13-oxidation. In contrast to our previous tetranor dihydro 15-keto PGF. For 15-keto dihydro report [74], we found that the 15-PGDH product PGF20, the metabolites were tetranor dihydro 9,1 l-dihydroxy-6, 15-diketo-2,3-dinor PGF10 was PGF10 and tetranor dihydro l5-keto PGF10. It ap- 768 Sunet a!

64%

0 15-keto dihydro PGE2 +:ss:c;OH

10%

OH OH OH

63% + OH° OH0H 0 - 30% 15-Keto dihydro PGF20 50% + unknown - 20% Fig.6. Proposed metabolic pathways of 12,14-dihydro-15-keto-PGE2 and /3,14-dihydro-15-keto-PGF2, in isolated perfused rabbit kidney. The stereo chemistry of reduction of C-9 or C-IS keto groups have not been determined. pears that both C-9 and C-iS keto groups are nonpolar lipids with greater water solubility so that susceptible to reduction. As we discussed in the they can be easily excreted by the urinary route. It previous section, the nonspecific type II PGDH can may also be used as a fatty acid for generating interconvert hydroxyi and keto functions at both metabolic energy. The physiologic roles of the type the C-9 and C-IS positions. I PGDH are puzzling. We hypothesize that the The metabolite pattern of PGF2 in rabbit kidney enzyme may be reserved for metabolic control of compared favorably with that obtained from rabbit intrarenal prostaglandin activities. The study of the urine after i.v. PGF2c. administration. Svanborg and fate of endogenous renal prostaglandins should give Bygdeman [55] reported that tetranor PGF1a, te- some insight into this important question. tranor dihydro PGF10, and tetranor dihydro 15-keto PGF1 counted for two thirds of recovered urinary Reprint requests to Dr. F. F. Sun, Department of Experimen- radioactivity from PGF2a-treated rabbit. In addi- tal Biology, The Upjohn Company, Kalamazoo, Michigan tion, the reduction of 9-keto groups of the E prosta- 49001, USA giandins in vivo has been reported in man [541.Only References PGI2 showed a trace of w-hydroxylation. Therefore, 1. LEE JB, CROWSHAW K, TAKMAN BH, ATTREP KA, w-hydroxylation of prostaglandins, if occurring at GOUGOUTAS JZ: Identification of prostaglandins E2, F2,, and all, must be of minor importance. A2 from rabbit kidney medulla. Biochem J 105:1251—1260, Summary. We found the kidney to be a major site 1967 2. VAN PRAAGD,FARBER SJ, STANLEY D: Leucotrienes as of prostaglandin metabolism. The circulating pros- lipoxygenase products of rabbit kidney. Fed Proc 39:1897, taglandins in the vascular compartment are exten- 1980 sively graded by the mitochondrial 13-oxidation sys- 3. Nisi-iiaoiu T, MATSUOKA Y, MATSUMOTO T: Studies on tem to shorter chain compounds. The keto groups absorption, distribution, excretion and retabolism of 1 113- at C-9 and C-IS are reduced to alcohol. The 15- H prostaglandin E2 in rats. Pharmacometrics 8:797—805, 1974 PGDH plays only a minor role in the handling of 4. DUNN MJ, HOOD VL: Prostaglandins and the kidney. Am J circulating prostaglandins. The purpose for exten- Physiol 233:F169—F184, 1977 sive renal degradation is probably to provide these 5. WEBER PC, SCHERER B, SiEss W, HELD E, SCHNERMANN J: Renal metabolism of prostaglandins 769

Formation and action of prostaglandins in the kidney. Kim the prostaglandin forming cyclooxygenase in renal cortex. Wochenschr 57:1021—1029, 1979 AmJ Physiol 235:F451—F457,1978 6. FLAMENBAUM W, KLEINMAN JG: Prostaglandins and renal 25. SMITH WL, BELL TG, NEEDLEMAN P: Increased renal function, or "a trip down the rabbit hole," Chapter 9, in The tubular synthesis of prostaglandins in the rabbit kidney in Prostaglandins, edited by RAMWELL PW, New York, Plen- response to ureteral obstruction. Prostagiandins 18:269—277, um Press, 1977, p 267 1979 7. HAMBERG M: Biosynthesis of prostaglandins in the renal 26. ZUSMAN RM, KEISER HR: Prostaglandin biosynthesis by medulla of rabbit. FEBSLetters5:127—130, 1969 rabbit renomedullary interstitial cells in tissue culture. J Clin 8. BOHMAN S, LAR550N C: Prostaglandin synthesis in mem- Invest60:215—223,1977 brane fractions from the rabbit renal medulla. Acta Physiol 27. DUNN MJ, STALEY RS, HARRISON M: Characterization of Scand 94:244—258, 1975 prostaglandin production in tissue culture of rat renal medul- 9. PACE ASCIAK C, NASHAT M: Catabolism of an isolated, lary cells. Prostaglandmns 12:37—47, 1976 purified intermediate of prostaglandin biosynthesis by re- 28. TERRAGNO NA, MCGIFF JC, TERRAGNO A: Prostacyclin gions of the adult rat kidney. Biochim Biophys Acta 388:243— production by renal blood vessels. Clin Res 26:545A, 1978 253, 1975 29. BOHMAN 5: Demonstration of prostaglandin synthesis in 10. BLACKWELL GJ, FLOWER RJ, VANE JR: Some characteris- collecting duct cells and other cell types of rabbit renal tics of the prostaglandin synthesizing system in rabbit kidney medulla. Prostaglandins14:729—744,1977 microsomes. Biochim Biophys Acta 398:178, 1975 30. GRENIER FC, SMITH WL: Formation of 6-keto PGF,, by 11. PONG SS, LEVINE L: Biosynthesis of prostaglandins in collecting tubule cells isolated from rabbit renal papillae. rabbit renal cortex. Res Commun Chem Pathol Pharmacol Prostagiandins 16:759—772, 1978 13:115—123, 1976 31. HASSID A, KONIECZKOWSKI M, DUNN MJ: Prostaglandin 12. SCHWARTZMAN M, GAFINI Y, RAZ A: Properties of prosta- synthesis in isolated rat kidney glomeruli. Proc Nat! Acad glandin synthetase of rabbit kidney medulla. Eur J Biochem SciUSA 76:1155—1159, 1979 64:527—534, 1976 32. SRAER J, SRAER JD, CHANSEL D, RUSSO-MARIE F, 13. TAI HH, TA! CL, HOLLANDER CS: Biosynthesis of prosta- KOUZNETZOVA B, ARDAILLON R: Prostaglandin synthesis glandins in rabbit kidney medulla. Biochem J 154:257—264, by isolated rat renal glomeruli. Mo! Cell Endocrinoi 16:29— 1976 37, 1979 14. BHAT SG, YOSHIMOTO T, YAMAMOTO S, HAYASHI 0: 33. FOLKERT VW, SCHLONDORFF D: Prostaglandin synthesis in Solubilization and partial purification of prostaglandin endo- isolated glomeruli. Prostagiandins 17:79—86, 1979 peroxide synthetase of rabbit kidney medulla. Biochim 34. SRAER J, FOIDART J, MAHIEU P, ARDAILLON R: Prostaglan- Biophys Acta 529:398—408, 1978 din synthesis by glomeruli cultured cells. Kidney mt 16:839, iS. WHORTON AR, SMIGEL M, OATES JA, FROLICH JC: Region- 1979 al differences in prostacyclin formation by the kidney. 35. PETRULIES AS, AIKAWA M, DUNN MJ: Prostaglandin and Biochim Biophys Acta 529:176—180, 1978 thromboxane synthesis by rat glomerular epithelial cells. 16. HA5SID A, DUNN MJ: Microsomal prostaglandin biosynthe- Kidneymt16:858, 1979 sis of human kidney. JBiol Chem 255:2472—2475,1980 36. ZENSERTV, HERMAN CA, DAVIS BB: Effects of calcium and 17. DANON A, CHANG LCT, SWEETMAN BJ, NIES AS, OATES A23 187 on renal inner medullary prostaglandin E2 synthesis. JA: Synthesis of prostaglandins by rat renal papilla in vitro. Am J Physiol 238:E37l—E376, 1980 Biochim Biophys Acta 388:71—83, 1975 37. SILBERBAUER K, SINZINGER H: Cortex and medulla of rat 18. ZUSMAN RM, KEISER HR: Prostaglandin E2 biosynthesis by kidney generate different amounts of PGI2-like activity. rabbit renomedulla interstitial cells in tissue culture. J Biol ThrombRes13:1111—1118,1978 Chem252:2069—2071,1977 38. OLIw E, LUNDEN I, SJOQUIsT B, ANGGARD E: Determina- 19. ISAKSON PC, RAZ A, DENNY SE, WYCHE A, NEEDLEMAN P: tion of 6-keto PGF1,, in rabbit kidney and urine and its Hormonal stimulation of arachidonic release from isolated relation to sodium balance. Acta Physiol Scand 105:359—366, perfused organs: Relationship to prostaglandin biosynthesis. 1979 Prostaglandins14:853—869,1977 39. SUN FF, CHAPMAN JP, MCGUIRE JC: Metabolism of prosta- 20. HSUEI-I W, NEEDLEMAN P: Sites of lipase activation and glandin endoperoxide in animal tissues. Prostaglandins prostaglandin synthesis in isolated perfused rabbit hearts 14:1055—1074, 1977 and hydronephrotic kidneys. Prostaglandins 16:661—682, 40. OGINO N, MIYAMOTO T, YAMAMOTO 5, HAYAISHI 0: 1978 Prostaglandin endoperoxide E isomerase from bovine vesic- 21. NEEDLEMAN P, WYCHE A, BR0N50N SD, H0LMBERG S, ular gland microsomes, a glutathione requiring enzyme. J MORRISON AR: Specific regulation of peptide induced renal BioiChem 252:890—895, 1977 Biol Chem 254:9772—9777, prostaglandin biosynthesis. J 41. PORTER NA, WOLF RA, PAGELS WR, MAMATT U: A test 1979 22. DUNN MJ, LIARD iF, Diy F: Basal and stimulated rates of for the intermediary of 1i-HPETE in prostaglandin biosyn- renal secretion and excretion of prostaglandins E2, F2,, and thesis. Biochem Biophys Res Commun 92:349—355, 1980 l3,i4-dihydro-15-keto-PGF,, in the dog. Kidney mt 13:136— 42. LARSSON C, ANGGARD F: Regional differences in the forma- 143, 1978 tion and metabolism of prostaglandins in rabbit kidney. Eur J 23. SMITH WL, WILKIN GP: Immunochemistry of prostaglandin Pharmacoi21:30—36,1973 endoperoxide forming cyclooxygenases: the detection of the 43. NISSEN HM, ANDERSEN H: On the localization of prosta- cyclooxygenases in rat, rabbit and guinea pig kidneys by glandin dehydrogenase activity in the kidney. Histochemie immunofluorescence. Prostagiandins 13:873—892, 1977 14:189—200, 1968 24. SMITH WL, BELL TG: Immunohistochemical localization of 44. WOHLARB F, FEUSTEL A: On the histochemistry of prosta- 770 Sunet a!

glandin dehydrogenase. Ada Biol Med Germ 37:897—899, activity in the adult rat kidney. J Biol Chem 250:2789—2794, 1978 1975 45. WRIGHT iT, CORDER CN, TAYLOR R: Studies on rat kidney 60. MOORE PK, HOULT JRS: Prostaglandin metabolism in rabbit 15-hydroxy prostaglandin dehydrogenase. Biochem Phar- kidney identification and properties of a novel prostaglandin macol 25:1669—1673, 1976 dehydrogenase. Biochim Biophys Acta 528:276—287, 1978 46. HASSID A, LEVINEL:Multiple molecular forms of prosta- 61. WONG PY-K, LEE WH, REI5s RF, MCGIFF JC: Metabolism glandin 15-hydroxydehydrogenase and 9-keto reductase in of prostacyclin by a purified 9-hydroxy prostaglandin dehy- chicken kidney. Prostaglandins 13:503—516, 1977 drogenase of rabbit liver and human platelets. Fed Proc 47. TA! HH, TA! CL, HOLLANDER CS: Regulation of prostaglan- 39:392, 1980 din metabolism inhibition of 15-hydroxy prostaglandin dehy- 62. ELLIN A, ORRENIUS 0: Fatty acid hydroxylation in rat drogenase by thyroid hormones. Biochem Biophys Res Com- kidney cortex microsomes. Mo! Cell Biochem 8:69—79, 1975 mun 57:457—462, 1974 63. GRANSTROM E: Metabolism of prostaglandin F2, in swine 48. ARMSTRONG JM, BLACKWELL GJ, FLOWER Ri, MCGIFF JC, kidney. Biochem Biophys Ada 239:120—125, 1971 MULLANE KM, VANE JR: Genetic hypertension in rats is 64. HOULT JRS, MOORE PK: Pathways of prostaglandin F2 accompanied by a defect in renal prostaglandin catabolism. metabolism in mammalian kidneys. BrfPharmacol6l:615— Nature 260:582—586, 1976 626, 1977 49. BLACKWELL GJ, FLOWER RJ, VANE JR: Rapid reduction of 65. MOORE PK, HOULT iRS: Distribution of four prostaglandin prostaglandin 15-hydroxy dehydrogenase activity in rat tis- metabolizing enzymes in organs of the rabbit. Biochem sues after treatment with protein synthesis inhibitors. Br J Pharmacol 27:1839—1842, 1978 Pharmacol 55:233—238, 1975 66. RENNICK BR: Renal tubular transport of prostaglandins: 50. CHANG DG, TA! HH: Prostaglandin 9-keto reductase from Inhibition of probenecid and indomethacin. Am J Physiol swine kidney. Fed Proc 39:1857, 1980 233:F133—F137, 1977 51. LEE SC, PONG SS, KATZEN D, Wu KY, LEVINE L: Distri- 67. BIT0 LZ, BAROODY RA: Comparison of renal prostaglandin bution of prostaglandin F 9-keto reductase and type I and and p-aminohippuric acid transport process. Am J Physiol type II 15-hydroxy prostaglandin dehydrogenase in swine 234:F80—F88, 1978 kidney medulla and cortex. Biochemistry 14:142—145, 1975 68. IRIsH JM III: Secretion of prostaglandin E2 by rabbit proxi- 52. STONE KJ and HART M: Prostaglandin F2 9-keto reductase mal tubules. Am J Physiol 237:F268—F273, 1979 in rabbit kidney. Prostaglandins 10:273—279, 1975 69. KAUKER ML: Tracer microinjection studies of prostaglandin 53. Oiw E, LUNDEN1,ANGGARD E: Affinity chromatography E2 in the rat nephron. J Pharm Exp Ther 193:271—280, 1975 of 15-hydroxyprostaglandin dehydrogenases from swine kid- 70. FROLICH JC, WILSON TW, SWEETMAN BJ, SMIGEL JM, ney. Adv Prostaglandin Thromboxane Res 1:147—151, 1976 NIE5AS,CARR K, WATSON JT, OATE5 JA: Urinary prosta- 54. HAMBERG M, WILSON M: Structures of new metabolites of glandins: Identification and origin. J Clin Invest 55:763—770, prostaglandin E2 in man, in Advances in Biosciences, 1975 Vieweg, Pergamon Press, 1973, vol. 9, pp. 39—48 71. WILLIAMS WM, FROLICH JC, NIES AS, OATES JA: Urinary 55. SVENBORG K, BYGDEMAN M: Metabolism of prostaglandin prostaglandin site of entry into renal tubular fluid. Kidney mt F2, in rabbit EuR J BIOCHEM 28:127—135, 1972 11:256—260, 1977 56. WEBER PC, LARSSON C, SCHERER B: Prostaglandin E2 9- 72. SCHUREK Hi, BRECHT JP, LOHFERT H, HIERHALZER K: The keto reductase as a mediator of salt intake related prosta- basic requirements for the function of the isolated cell free glandin renin interaction. Nature 266:65—66, 1977 perfused rat kidney. Pfluegers Arch 354:349—365, 1975 57. HANSENHS:Purification and characterization of a 15-keto 73. SCHOLZ R, THURMAN RG, WILLIAMSON JR, CHANCE B, prostaglandin A'3 reductase from the bovine lung. Biochim BUCHER T: Flavin and pyridine nucleotide oxidation reduc- BiophysActa 574:136—145, 1979 tion changes in perfused rat liver. J Biol Chem 244:2317— 58. WESTBROOK C, JARABAK J: 15-Keto prostaglandin A'3 re- 2324, 1969 ductase from human placenta: Purification, kinetics and 74. WONG PY-K, MCGIFF JC, CAGAN L, MALIK KU, SUN FF: inhibitor binding. Arch Biochem Biophys 185:429—442, 1978 Metabolism of prostacyclin in the rabbit kidney. JBiol Chem 59. PACE A5cIAK C: Prostaglandin 9-hydroxy dehydrogenase 254:12—14, 1979