Demonstration of prostaglandin synthesis in collecting duct cells and other cell types of the rabbit renal medulla.

The renal medulla has a high capacity for prostaglandin production and the interstitial cells, which contain abundant lipid inclusions have been suggested to be the site of synthesis. However, histochemical studies have indicated that the collecting ducts are the main site of production. The object of the present study was to study the distribution of prostaglandin synthetase in the rabbit renal medulla by direct, quantitative determination of the enzyme activity in different cellular fractions. Slices were cut from rabbit renal papilla and immersed in a hypertonic saline solution. 92% of the collecting duct cells were then removed from the slices by suction through a micropipette. The remaining dissected slices thus contained mainly three cell types, cells of Henle's loop, endothelial cells, and interstitial cells. The isolated collecting duct fraction, the corresponding dissected slices, from which the colelcting duct cells were removed, as well as intact slices were assayed for prostaglandin synthetase activity using a quantitative assay with [14C] arachidonate as substrate. Of the prostaglandin in synthetase activity 39% was found in the collecting ducts, 53% in the dissected slices, and 7% in the dissection medium. It is thus concluded that significant prostaglandin synthetase activity is present in collecting duct cells as well as in at least one other cell type of the medulla.     

Enhancement of renal medulla prostaglandin synthetase activity by dexamethasone treatment in the rat

We studied in rats the effect of dexamethasone (2.5 mg/kg per week) on the conversion of radiolabeled arachidonic acid to prostaglandins by renal medulla slices, microsomes, and homogenates. The steroid did not affect the rate of conversion of arachidonic acid to prostaglandins by renal medulla slices, but significantly increased the rate of conversion by both the microsomes and the 10,000 × g supernatatant of renal medulla homogenates. We conclude (a) that dexamethasone treatment increases the activity of renal medulla prostaglandin synthetase measured in broken cells preparations, and (b) that such a change in enzyme activity is not manifested by augmentation of prostaglandin synthesis in renal medulla slices incubated with exogenous arachidonic acid.     

Immunochemistry of prostaglandin endoperoxide-forming cyclooxygenases: the detection of the cyclooxygenases in rat, rabbit, and guinea pig kidneys by immunofluorescence.

Rabbit antiserum has been prepared against the prostaglandin endoperoxide-forming cyclooxygenase (EC 1.14.99.1) purified from sheep vesicular glands. Ouchterlony double diffusion and immunoelectrophoretic analyses indicate that the anti-cyclooxygenase serum is monospecific for the enzyme. The anti-cyclooxygenase serum reacts with both active and inactivated forms of the sheep vesicular gland (SVG) cyclooxygenase. Furthermore, the immune serum precipitates solubilized microsomal cyclooxygenases from each of six other tissues examined, including bovine seminal vesicle, rabbit kidney medulla, guinea pig lung, dog spleen, sheep uterus, and human platelets. Anti-SVG cyclooxygenase serum was used in combination with fluorescein isothiocyanate )FITC)-labeled goat anti-rabbit IgG to detect cyclooxygenases in cryostat sections from rat, rabbit and guinea pig kidneys by immunofluorescence. Highly prominent fluorescence was associated only with the epithelial cells lining the collecting ducts in rabbit and guinea pig kidneys, and except for the nucleus, was uniformly distributed within the interior of these cells. In rat kidney, fluorescence was detected not only in collecting tubules but also in the interstitial cells of the renal papilla. Our results are consistent with the emerging hypothesis that PGE2 produced intrarenally plays a physiological role in natriuresis.     

Does diaminobenzidine demonstrate prostaglandin synthetase?

Summary A method histochemical localization of prostaglandin synthetase using DAB, potassium cyanide and polyunsaturated fatty acid has been revised. The arachidonic acid-induced DAB oxidation observed in the secretory epithelium of sheep vesicular glands and in collecting tubules as well as interstitial cells of rabbit kidney medulla was found to be insensitive to antiinflammatory cyclooxygenase (formerly referred as prostaglandin synthetase) inhibitors, such as indomethacin, aspirin, mefenamic acid and paracetamol, whereas aminotriazole caused complete inhibition of the reaction. Furthermore, DAB was oxidized in the presence of polyunsaturated fatty acids inconvertible to prostaglandins (linoleic and linolenic acid) as well as in the presence of H₂O₂ — in the latter case reaction possessed identical features with that induced by fatty acids. Ultrastructurally, the reaction product was localized on the membranes of nuclear envelope and endoplasmic reticulum. On the ground of the results obtained a hypothesis is presented, that the polyunsaturated fatty acid-induced DAB oxidation is due to a peroxidatic activity of the investigated tissues. Possible relations between such peroxidatic activity and prostaglandin biosynthesis are discussed.     

Prostaglandin H synthase immunoreactivity localized by immunoperoxidase technique (PAP) in the small intestine and kidney of rabbit and guinea-pig.

Prostaglandins and inhibitors of prostaglandin synthesis have striking regulatory effects on intestinal muscularis externa. We suggested earlier that a population of macrophage-like cells, located between the external muscle layers might release prostaglandins with a local effect on enveloping interstitial cells of Cajal, postulated pacemaker cells of the gut. To determine cellular production site(s) of prostaglandin we applied monoclonal antibodies against prostaglandin H synthase combined with the PAP technique to sections of rabbit and guinea-pig small intestine and kidney. In rabbit small intestine muscle cells in the circular muscle layer and in the muscularis mucosae were positive, longitudinal muscle negative. Vascular endothelial cells and serosal mesothelial cells were stained. In guinea-pig all muscle layers were unstained but endothelial and mesothelial cells were stained together with unidentified cells in the outermost submucosa. In rabbit kidney, positive staining of collecting ducts, interstitial cells, the parietal layer of Bowman's capsule and arterial endothelial cells was present. Furthermore, we found prostaglandin synthase antigenicity in the epithelial cells lining the loop of Henle, not described before. In guinea-pig medullary collecting ducts were stained and the papilla was lined by stained epithelial cells. The results show a species variation in the distribution of recognizable levels of prostaglandin H synthase. The impressive reaction in the mesothelium must be considered, when enzyme distribution is examined biochemically with fractionated tissue. Our findings do not support our hypothesis that macrophage-like cells are more potent sources of prostaglandins than smooth muscle cells.     

Immunochemistry of prostaglandin endoperoxide-forming cyclooxygenases: The detection of the cyclooxygenases in rat, rabbit, and guinea pig kidneys by immunofluorescence

Rabbit antiserum has been prepared against the prostaglandin endoperoxide-forming cyclooxygenase (EC 1.14.99.1) purified from sheep vesicular glands. Ouchterlony doùble diffusion and immunoelectrophoretic analyses indicate that the anti-cyclooxygenase serum is monospecific for the enzyme. The anti-cyclooxygenase serum reacts with both active and inactivated forms of the sheep vesicular gland (SVG) cyclooxygenase. Furthermore, the immune serum precipitates solubilized microsomal cyclooxygenase from each of six other tissues examined, including bovine seminal vesicles, rabbit kidney medulla, guinea pig lung, dog spleen, sheep uterus, and human platelets.Anti-SVG cyclooxygenase serum was used in combination with fluoresence isothiocyanate (FITC)-labelled goat anti-rabbit IgG to detect cyclooxygenase in cryostat sections from rat, rabbit and guinea pig kidneys by immunofluorescence. Highly prominent fluorescence was associated only with the epithelial cells lining the collecting ducts in rabbit and guinea pig kidneys, and except for the nucleus, was uniformly distributed within the interior of these cells. In rat kidney, fluorescence was detected not only in collecting tubules but also in the interstitial cells of the renal papilla. Our results are consistent with the emerging hypothesis that PGE₂ produced intrarenally plays a physiological role in natriuresis.     

Prostaglandin H synthase immunoreactivity localized by immunoperoxidase technique (PAP) in the small intestine and kidney of rabbit and guinea-pig

Summary Prostaglandins and inhibitors of prostaglandin synthesis have striking regulatory effects on intestinal muscularis externa. We suggested earlier that a population of macrophage-like cells, located between the external muscle layers might release prostaglandins with a local effect on enveloping interstitial cells of Cajal, postulated pacemaker cells of the gut.To determine cellular production site(s) of prostaglandin we applied monoclonal antibodies against prostaglandin H synthase combined with the PAP technique to sections of rabbit and guinea-pig small intestine and kidney. In rabbit small intestine muscle cells in the circular muscle layer and in the muscularis mucosae were positive, longitudinal muscle negative. Vascular endothelial cells and serosal mesothelial cells were stained. In guinea-pig all muscle layers were unstained but endothelial and mesothelial cells were stained together with unidentified cells in the outermost submucosa. In rabbit kidney, positive staining of collecting ducts, interstitial cells, the parietal layer of Bowman's capsule and arterial endothelial cells was present. Furthermore, we found prostaglandin synthase antigenicity in the epithelial cells lining the loop of Henle, not described before. In guinea-pig medullary collecting ducts were stained and the papilla was lined by stained epithelial cells.The results show a species variation in the distribution of recognizable levels of prostaglandin H synthase. The impressive reaction in the mesothelium must be considered, when enzyme distribution is examined biochemically with fractionated tissue. Our findings do not support our hypothesis that macrophage-like cells are more potent sources of prostaglandins than smooth muscle cells.     

Work in progress correlation of renal medullary prostaglandin content and renal interstitial cell lipid droplets

Indomethacin administration and hydronephrosis in rabbits has been found to produce increases in the number and changes in the composition of the lipid droplets in the renal medullary interstitial cells. The response to indomethacin, a prostaglandin synthetase inhibitor, was dose dependent.Work is in progress to assess the effects of other non-steroidal antiinflammatory drugs on the renal inner medulla and the interstitial cell lipid droplets.     

Immunocytochemical localization of a renal glycoprotein (gpCDI) synthesized by cultured collecting duct cells

A sulfated, proline-rich glycoprotein (gpCDI, apparent molecular weight 200,000 in column chromatography and 150,000 in SDS-PAGE) was isolated from cultured renal collecting duct epithelium by centrifugation. Triton X100 extraction and DEAE-cellulose ion exchange chromatography. A DEAE-cellulose ion exchange chromatography fraction with the enriched gpCDI was used for immunization of guinea pigs. The antiserum was prepared for antigen localization by indirect immunofluorescence in collecting duct cell cultures and in tissue sections of neonatal and adult rabbit kidneys. In the cultured collecting duct epithelium, antibody staining of the epithelium and structures of the extracellular matrix was age dependent. Cultures of dedifferentiated collecting duct monolayers revealed positive reaction in the cytoplasm. In neonatal and adult rabbit kidneys, the antibody was localized in the entire collecting duct system but not in the collecting duct ampullae of the newborn kidney. Staining of the cytoplasm was found only in medullary collecting ducts of the neonatal kidney; other portions revealed staining mostly at the basal circumference of the tubule and at the luminal cell borders. Apart from collecting ducts, no other tubular segments were reactive. The cortical and the medullary interstitium contained fluorescent fibres which were concentrated around vascular structures. A possible relation between gpCDI and collagenous compounds is discussed. Bowman's capsule reacted positively, whereas staining of the mesangial matrix was weak. The localization of the antigen, as revealed by indirect immunofluorescence, suggests that gpCDI occurs both in intracellular and extracellular (interstitial) location. Two main points are emphasized: Firstly gpCDI is considered an important constituent in different stages of collecting duct development, and secondly, the staining pattern of the antibody varies with the different portions of both young and adult kidney collecting ducts; this staining heterogeneity may correspond with the known regional differences of collecting duct functions.     

Immunohistochemical localization of a protein fraction derived from rabbit renal papilla

Studies were carried out to define antigenic characteristics of the rabbit renal collecting duct. Renal papillae of adult rabbits were homogenized, centrifuged, and the 600 X g pellet was extracted with 0.5% Triton X-100 in the presence of 1 M NaCl. The crude extract was fractionated on an anion exchange column (DEAE cellulose). A fraction enriched in acidic proteins that co-purified with a radioactive 150 kd glycoprotein from cultured collecting duct cells (Minuth 1982), was used for immunization of guinea pigs. The antiserum shows the following characteristics as revealed by indirect immunofluorescence on the rabbit kidney: 1) Among all tubular epithelial cells only principal cells of the collecting duct and the connecting tubule cell show immunoreactivity. 2) The antiserum decorates the epithelial-interstitial interface of the whole collecting duct as well as of connecting tubule and thick ascending limb of Henle's loop. 3) There is immunoreactivity of interstitial fibers throughout the kidney. 4) Epithelial cells in a variety of other organs in rabbit did not react with the antiserum. Our data demonstrate an antigenic distinction of both, the connecting tubule cell and the principal cell, discriminating these cells from other tubular epithelial cells including the intercalated cells of the collecting duct system. Furthermore, our findings point to a heterogeneity along the distal nephron with respect to the constituents of the epithelial-interstitial interface.     

Pathways for organic osmolyte synthesis in rabbit renal papillary tissue, a metabolic study using 13C-labeled substrates

Renal papillary collecting duct cells have been postulated to adapt their intracellular osmolality to the large changes in interstitial osmolality by changing their content of 'non-perturbing' organic osmolytes such as sorbitol and myo-inositol. 13C-NMR was used in this study to elucidate the metabolic pathways leading to a synthesis of those compounds. Incubation of rabbit renal papillary tissue with [1-13C]glucose showed label scrambling mainly into sorbitol (C-1) and lactate (C-3). This result confirms activity of aldose reductase and glycolytic enzymes in renal papillary cells. Using [3-13C]alanine or [2-13C]pyruvate as carbon source, 13C-labeling of sorbitol and myo-inositol was observed, indicating that renal papillary tissue possesses, in addition, gluconeogenic activity. The latter assumption is supported by the result that in enzyme assays rabbit kidney papilla and isolated rat kidney papillary collecting duct cells show significant fructose-1,6-bisphosphatase activity.     

Immunocytochemical localization of a renal glycoprotein (gp CD I) synthesized by cultured collecting duct cells

Summary A sulfated, proline-rich glycoprotein (gp_CDI, apparent molecular weight 200,000 in column chromatography and 150,000 in SDS-PAGE) was isolated from cultured renal collecting duct epithelium by centrifugation, Triton X100 extraction and DEAE-cellulose ion exchange chromatography. A DEAE-cellulose ion exchange chromatography fraction with the enriched gp_CDI was used for immunization of guinea pigs. The antiserum was prepared for antigen localization by indirect immunofluorescence in collecting duct cell cultures and in tissue sections of neonatal and adult rabbit kidneys. In the cultured collecting duct epithelium, antibody staining of the epithelium and structures of the extracellular matrix was age dependent. Cultures of dedifferentiated collecting duct monolayers revealed positive reaction in the cytoplasm. In neonatal and adult rabbit kidneys, the antibody was localized in the entire collecting duct system but not in the collecting duct ampullae of the newborn kidney. Staining of the cytoplasm was found only in medullary collecting ducts of the neonatal kidney; other portions revealed staining mostly at the basal circumference of the tubule and at the luminal cell borders. Apart from collecting ducts, no other tubular segments were reactive. The cortical and the medullary interstitium contained fluorescent fibres which were concentrated around vascular structures. A possible relation between gp_CDI and collagenous compounds is discussed. Bowman's capsule reacted positively, whereas staining of the mesangial matrix was weak. The localization of the antigen, as revealed by indirect immunofluorescence, suggests that gp_CDI occurs both in intracellular and extracellular (interstitial) location. Two main points are emphasized: Firstly, gp_CDI is considered an important constituent in different stages of collecting duct development, and secondly, the staining pattern of the antibody varies with the different portions of both young and adult kidney collecting ducts; this staining heterogeneity may correspond with the known regional differences of collecting duct functions.     

Does diaminobenzidine demonstrate prostaglandin synthetase? A study on polyunsaturated fatty acid-induced DAB oxidation in sheep vesicular glands and rabbit kidney medulla.

A method histochemical localization of prostaglandin synthetase using DAB, potassium cyanide and polyunsaturated fatty acid has been revised. The arachidonic acid-induced DAB oxidation observed in the secretory epithelium of sheep vesicular glands and in collecting tubules as well as intersititial cells of rabbit kidney medulla was found to be insensitive to antiinflammatory cyclooxygenase (formerly referred as prostaglandin synthetase) inhibitors, such as indomethacin, aspirin, mefenamic acid and paracetamol, whereas aminotriazole caused complete inhibition of the reaction. Furthermore, DAB was oxidized in the presence of polyunsaturated fatty acids inconvertible to prostaglandins (linoleic and linolenic acid) as well as in the presence of H2O2--in the latter case reaction possessed identical features with that induced by fatty acids. Ultrastructurally, the reaction product was localized on the membranes of nuclear envelope and endoplasmic reticulum. On the ground of the results obtained a hypothesis is presented, that the polyunsaturated fatty acid-induced DAB oxidation is due to a peroxidatic activity of the investigated tissues. Possible relations between such peroxidatic activity and prostaglandin biosynthesis are discussed.     

Immunohistochemical localization of a protein fraction derived from rabbit renal papilla

Summary Studies were carried out to define antigenic characteristics of the rabbit renal collecting duct. Renal papillae of adult rabbits were homogenized, centrifuged, and the 600×g pellet was extracted with 0.5% Triton X-100 in the presence of 1 M NaCl. The crude extract was fractionated on an anion exchange column (DEAE cellulose). A fraction enriched in acidic proteins that co-purified with a radioactive 150 kd glycoprotein from cultured collecting duct cells (Minuth 1982), was used for immunization of guinea pigs. The antiserum shows the following characteristics as revealed by indirect immunofluorescence on the rabbit kidney: 1) Among all tubular epithelial cells only principal cells of the collecting duct and the connecting tubule cell show immunoreactivity. 2) The antiserum decorates the epithelial-interstitial interface of the whole collecting duct as well as of connecting tubule and thick ascending limb of Henle's loop. 3) There is immunoreactivity of interstitial fibers throughout the kidney. 4) Epithelial cells in a variety of other organs in rabbit did not react with the antiserum.Our data demonstrate an antigenic distinction of both, the connecting tubule cell and the principal cell, discriminating these cells from other tubular epithelial cells including the intercalated cells of the collecting duct system. Furthermore, our findings point to a heterogeneity along the distal nephron with respect to the constituents of the epithelial-interstitial interface.     

Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin on hepatic and renal prostaglandin synthetase

This study examines the possibility that the very toxic compound, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), produces its toxic effects through induction or repression of microsomal prostaglandin synthetase (cyclooxygenase). The effects of TCDD on microsomal synthesis of prostaglandin from [¹⁴C]arachidonic acid in rabbit liver and kidney medulla were examined 24 and 72 hr after TCDD administration. A hepatotoxic dose of TCDD (30 μg/kg) did not affect prostaglandin synthetase activity of rabbit liver or kidney medulla microsomes at either time point, although other microsomal enzymes (cytochrome P-488) were altered in both tissues.     

Paracrine regulation of the epithelial Na+ channel in the mammalian collecting duct by purinergic P2Y2 receptor tone

Growing evidence implicates a key role for extracellular nucleotides in cellular regulation, including of ion channels and renal function, but the mechanisms for such actions are inadequately defined. We investigated purinergic regulation of the epithelial Na+ channel (ENaC) in mammalian collecting duct. We find that ATP decreases ENaC activity in both mouse and rat collecting duct principal cells. ATP and other nucleotides, including UTP, decrease ENaC activity via apical P2Y2 receptors. ENaC in collecting ducts isolated from mice lacking this receptor have blunted responses to ATP. P2Y2 couples to ENaC via PLC; direct activation of PLC mimics ATP action. Tonic regulation of ENaC in the collecting duct occurs via locally released ATP; scavenging endogenous ATP and inhibiting P2 receptors, in the absence of other stimuli, rapidly increases ENaC activity. Moreover, ENaC has greater resting activity in collecting ducts from P2Y2-/- mice. Loss of collecting duct P2Y2 receptors in the knock-out mouse is the primary defect leading to increased ENaC activity based on the ability of direct PLC stimulation to decrease ENaC activity in collecting ducts from P2Y2-/- mice in a manner similar to ATP in collecting ducts from wild-type mice. These findings demonstrate that locally released ATP acts in an autocrine/paracrine manner to tonically regulate ENaC in mammalian collecting duct. Loss of this intrinsic regulation leads to ENaC hyperactivity and contributes to hypertension that occurs in P2Y2 receptor-/- mice. P2Y2 receptor activation by nucleotides thus provides physiologically important regulation of ENaC and electrolyte handling in mammalian kidney.     

L-Arginine uptake affects nitric oxide production and blood flow in the renal medulla

Experiments were performed to determine whether L-arginine transport regulates nitric oxide (NO) production and hemodynamics in the renal medulla. The effects of renal medullary interstitial infusion of cationic amino acids, which compete with L-arginine for cellular uptake, on NO levels and blood flow in the medulla were examined in anesthetized rats. NO concentration in the renal inner medulla, measured with a microdialysis-oxyhemoglobin trapping technique, was significantly decreased by 26-44% and renal medullary blood flow, measured by laser Doppler flowmetry, was significantly reduced by 20-24% during the acute renal medullary interstitial infusion of L-ornithine, L-lysine, and L-homoarginine (1 micromol.kg(-1).min(-1) each; n = 6-8/group). In contrast, intramedullary infusion of L-arginine increased NO concentration and medullary blood flow. Flow cytometry experiments with 4-amino-5-methylamino-2',7'-difluorescein diacetate, a fluorophore reactive to intracellular NO, demonstrated that L-ornithine, L-lysine, and L-homoarginine decreased NO by 54-57% of control, whereas L-arginine increased NO by 21% in freshly isolated inner medullary cells (1 mmol/l each, n > 1,000 cells/experiment). The mRNA for the cationic amino acid transporter-1 was predominantly expressed in the inner medulla, and cationic amino acid transporter-1 protein was localized by immunohistochemistry to the collecting ducts and vasa recta in the inner medulla. These results suggest that L-arginine transport by cationic amino acid transport mechanisms is important in the production of NO and maintenance of blood flow in the renal medulla.     

Morphology of rabbit collecting duct.

Recently the assumed structural and functional homogeneity of the collecting duct (CD) has been questioned. The objective of this study was to determine if heterogeneity occurs in luminal surface membrane structure or in cytoplasmic configuration of cells in the collecting duct or both. Straight segments of cortical and medullary CD were examined in perfusion-fixed rabbit kidneys with scanning electron microscopy (SEM), light (LM) and transmission electron microscopy (TEM). Principal cells were the most abundant cells in all CD regions; intercalated cells comprised 37% of the cell population on the cortex, 18% in the outer medulla, and less than 1% in the inner medulla. SEM revealed two surface patterns among the ciliated principal cells: 1, located in the cortex and outer medulla, with few surface microvilli, and 2, located in the inner medulla, with abundant microvilli. Intercalated cells exhibited four distinctive luminal surface configurations: I, numerous short microvilli; II, both short and elongate microvilli; III, microplicae alone; and IV, both microvilli and microplicae. Intercalated cells with patterns I and II were predominant in the cortex, while cells with patterns III and IV were most common at the corticomedullary junction. TEM confirmed that marked variation existed in cytoplasmic structures of both principal and intercalated cells. These findings may either indicate the presence of several specific types of principal and intercalated cells or reflect different functional states of the principal and intercalated cells. Regardless of their significance, their presence must be considered in studies seeking to establish precise structural-functional relationships in this region of the rabbit renal tubule.     

Histochemical localization of enzyme activity in the kidneys of male marmosets (Callithrix jacchus and Callithrix penicillata).

The distribution of several hydrolases and oxidoreductases was studied in the renal parenchyma of adult male marmosets (Callithrix jacchus and Callithrix penicillata). The oxidative enzymes showed a high reactivity in the proximal and distal tubules, whereas the hydrolases reacted strongly in the proximal tubules but only weakly or not at all in the thick limb of Henle's loop, distal tubules and collecting ducts. The NAD-dependent enzymes (except alpha-GPDH) showed a stronger reactivity in the proximal tubules, while the NADP-dependent ones were more reactive in the thick limb of Henle's loop and distal convoluted tubules. Two groups of interstitial cells were found in the medulla. A first group inside the outer medulla, showing cells rich in acid phosphatase and nonspecific esterases and a second group, close to the papilla, reactive to a certain number of oxidative enzymes. A different reactivity in cells of the distal convoluted tubules, thick limb of Henle's loops and collecting ducts (dark cells) was seen in the case of some enzymes like nonspecific esterase, alpha-GPDH and SDH.     

Enriched prostaglandin E-9 ketoreductase activity in outer medullary cells of the rabbit kidney

PGE2 metabolism was examined in rabbit renal slices and cell suspensions from the outer medulla, enriched (TALH) and depleted (OMC) for the thick ascending limb of Henle's loop. Metabolism was negligible in intact cells, either OMC or TALH fractions. However, in OMC and TALH homogenates, transformation of PGE2 to PGF2 alpha by NADPH-dependent prostaglandin E-9 ketoreductase (PGE-9KR) was observed at a PGE2 concentration of 4 X 10(-9) M. This activity was not reversible and was enriched ten-fold in the TALH with 41% of PGE2 transformed to PGF2 alpha after 30 min incubation. PGF2 alpha formation from PGE2 could not be detected in homogenates of cortex, medulla or papilla. PGE-9KR activity, particularly in the thick ascending limb, may be a source of PGF2 alpha in urine.