Gene expression of prostanoid forming enzymes along the rat nephron

BACKGROUND: To obtain information about the general capability of nephron segments to elaborate prostanoids, we determined the gene expression of key enzymes for prostanoid formation. METHODS: For this goal mRNAs were assayed for cyclooxygenases-1 and -2 as well as for the synthases of prostaglandin D2 (PGD2), prostaglandin E2 (PGE2), prostacyclin (PGI2) and thromboxane A2 (TXA2) in microdissected rat nephron segments by RT-PCR. RESULTS: Cyclooxygenase-1 (COX-1) mRNA was strongly expressed in all segments of the collecting ducts and to a lesser extent in glomeruli. COX-2 mRNA was found in the cortical thick ascending limb of Henle, and weaker expression also was detected in glomeruli. The lipocalin-type PGD synthase mRNA displayed a broad expression pattern in the cortex and outer medulla, including proximal convoluted tubule, thick ascending limb of Henle, distal convoluted tubule, and cortical and outer medullary collecting duct. The hematopoietic PGD synthase mRNA was restricted to the outer medullary collecting duct, and the membrane-associated PGE-synthase mRNA was exclusively expressed in the whole collecting duct system. Prostacylin-synthase mRNA was found in the whole kidney, but not in any microdissected nephron segment analyzed in this study. TXA-synthase mRNA was expressed in glomeruli. CONCLUSION: Given that the existence of cyclooxygenase in combination with the different PG-synthases is a prerequisite for the formation of prostanoids, our data suggest that PGD2 is mainly formed in the thick ascending limb and in the collecting duct, while PGE2 appears to be mainly generated by the collecting ducts. Probably no formation of PGI2 occurs within the nephron. Whether TXA2 can be formed by nephron segments remains questionable.     

Expression of RhCG,a new putative NH(3)/NH(4)(+) transporter,along the rat nephron

Two non-erythroid members of the erythrocyte Rhesus (Rh) protein family, RhBG and RhCG, have been recently cloned in the kidney. These proteins share homologies with specific NH(3)/NH(4)(+) transporters (Mep/Amt) in primitive organisms and plants. When expressed in a Mep-deficient yeast, RhCG can function as a bidirectional NH(3)/NH(4)(+) transporter. The aim of this study was to determine the intrarenal and intracellular location of RhCG in rat kidney. RT-PCR on microdissected rat nephron segments demonstrated expression of mRNAs encoding RhCG in distal convoluted tubules, connecting ducts, and cortical and outer medullary collecting ducts but not in proximal tubules and thick ascending limbs of Henle's loop. Immunolocalization studies performed on rat kidney sections with rabbit anti-human RhCG 31 to 48 antibody showed labeling of the apical pole of tubular cells within the cortex, the outer medulla, and the upper portion of the inner medulla. All cells within connecting tubules had identical apical staining. In cortical collecting ducts, a subpopulation of cells that has either apical staining (alpha-intercalated cells) or diffuse staining (beta-intercalated cells) for the beta1 subunit of the H(+)-ATPase, was heavily stained at their apical pole with the RhCG antibody while principal cells identified as H(+)-ATPase negative cells showed a faint apical staining for RhCG that was much less intense than in adjacent intercalated cells. In the outer medulla and the upper portion of the inner medulla, RhCG labeling was restricted to a subpopulation of cells within the collecting duct that apically express the beta1 subunit of the H(+)-ATPase, indicating that RhCG expression in medullary collecting ducts is restricted to intercalated cells. No labeling was seen in glomeruli, proximal tubules, and limbs of Henle's loop. Immunoblotting of apical membrane fractions from rat kidney cortex with the rabbit anti-human RhCG 31 to 48 antibody revealed a doublet band at approximatively 65 kD.     

Role of EP2 and EP4 receptor-selective agonists of prostaglandin E(2) in acute and chronic kidney failure

We tested the efficacy of three selective agonists of prostaglandin E(2) (PGE(2)) receptor, EP2 (CP-536,745-01), EP2/4 (CP-043,305-02), and EP4 (CP-044,519-02), in two models of acute and chronic kidney failure. In the nephrotoxic mercury chloride (HgCl(2)) rat model of acute kidney failure systemically administered EP4 agonist reduced the serum creatinine values and increased the survival rate. Although the EP2 or the EP2/4 agonist did not change the serum creatinine values, the EP2 receptor agonist increased the survival rate. Histological evaluation of kidneys from EP4-treated rats indicated less proximal tubular necrosis and less apoptotic cells. In a rat model of chronic renal failure, the three receptor agonists decreased the serum creatinine and increased the glomerular filtration rate at 9 weeks following therapy. Kidneys treated with the EP4 agonist had less glomerular sclerosis, better preservation of proximal and distal tubules and blood vessels, increased convoluted epithelium proliferation and less apoptotic cells. Nephrectomy had no influence on the expression of the EP4 receptor, whereas EP2 receptor expression was reduced by 50% and then corrected following treatment with EP2 and EP2/4 receptor agonists. These findings suggest that PGE(2) has an important role in acute kidney failure via the EP4 receptor, whereas in chronic kidney failure both EP2 and EP4 receptors are equally important in preserving the progression of chronic kidney failure. Thus, agonism of EP2 and EP4 receptors may provide a basis for treating acute and chronic kidney failure.     

Bone biomechanical properties in prostaglandin EP1 and EP2 knockout mice

Prostaglandins play an important role in regulating the bone adaptation response to mechanical stimuli. Prostaglandin E2 (PGE2) is an effective modulator of bone metabolism. Administration of PGE2 to rodents results in increased cancellous and cortical bone mass translating into enhanced mechanical strength. The PGE2 influence on bone is mediated through four well-characterized receptors (EP1, EP2, EP3, and EP4). Although the PGE2 pathways and mechanisms of action on cells involved in bone adaptation are still under investigation, it is now known that each receptor plays a unique role in regulating PGE2-related bone cell function. The EP1 subtype is coupled with Ca2+ mobilization. The EP2 subtype stimulates cyclic adenosine monophosphate (cAMP) formation. cAMP in turn is responsible for the early cellular signal that stimulates bone formation. This study compared physical and biomechanical properties of bone in EP1 and EP2 knockout mice to their corresponding wild-type controls. Ash weight was measured in the ulnae, and femurs and vertebral bodies were tested in three-point bending and compression, respectively. The results suggest: (a) EP1 receptors have a minimal influence on skeletal strength or size in mice; and (b) EP2 receptors have a major influence on the biomechanical properties of bone in mice. The absence of EP2 receptors resulted in weak bone biomechanical strength properties in the EP2 knockout model as compared with the corresponding wild-type control mice.     

Prostaglandin E2 receptors EP2 and EP4 are down-regulated in human mononuclear cells after injury

BACKGROUND: Recent characterization of prostaglandin receptor subtypes shows that each is critical to cellular functions and operates through separate signaling pathways that may explain differing effects of prostanoids. This study aimed to determine whether prostaglandin receptors EP2 and EP4 are modulated after injury and to evaluate the effect of prostaglandin E(2) (PGE(2)) addition and blockade on EP receptor expression. METHODS: Peripheral blood mononuclear cells (PBMCs) isolated from 10 patients sustaining fracture or burn injury and 10 control subjects were stimulated with lipopolysaccharide +/- NS-398, an inhibitor of PGE(2) production. Samples were evaluated for production of PGE(2), tumor necrosis factor--alpha, and leukotriene B(4) as well as mRNA expression of EP receptors and COX-2. EP receptor expression was also evaluated after treating control PBMCs with PGE(2). RESULTS: PBMCs from injured patients exhibited significant increases in PGE(2) production and COX-2 mRNA compared with control subjects, and these increases were inhibited by NS-398. In contrast, EP2 and EP4 receptors were markedly down-regulated after injury and NS-398 restored expression to control levels. Decreased EP2 and EP4 receptor expression after injury was replicated by coincubation of PBMCs with PGE(2). CONCLUSIONS: Specific PGE(2) receptors are down-regulated after injury and NS-398 reverses this response. Furthermore, PGE(2) mediates EP2 and EP4 down-regulation. These data suggest that specific EP receptor subtypes may provide critical targets for augmenting the immune response after injury in humans.     

Decreased sensitivity of distal nephron and collecting duct to parathyroid hormone in pseudohypoparathyroidism type I

Parathyroid hormone (PTH) transiently increases urinary excretion of the lysosomal enzyme, N-acetyl-beta-D-glucosaminidase, which is distributed mainly in proximal tubules. The response is reduced in pseudohypoparathyroidism (PHP) type I, which is characterized by target-organ resistance to PTH. Evidenced by normal calcium resorption, distal tubule sensitivity to PTH has been believed to be normal in this disorder. This hypothesis was tested through a search for another marker of distal nephron sensitivity to PTH. In the human kidney, cathepsin D was expressed predominantly in distal segments of the nephron, cortical and medullary thick ascending limbs of Henle's loop, distal convoluted tubules, and connecting tubules and in cortical collecting ducts and medullary collecting ducts. PTH infusion transiently increased cathepsin D excretion in normal subjects. The cathepsin D response to PTH was reduced in the patients with PHP type I. The decrease in cathepsin D response in PHP type I indicates a resistance to PTH in the distal nephron (cortical thick ascending limbs of Henle's loop, distal convoluted tubules, and connecting tubules) and cortical collecting ducts. These observations suggest that the preservation of renal tubular sensitivity to PTH in this disorder may be confined to PTH-dependent calcium resorption in distal tubules.     

Cycloxygenase-2 is expressed in vasculature of normal and ischemic adult human kidney and is colocalized with vascular prostaglandin E2 EP4 receptors

The study was performed to elucidate the distribution and cellular localization of cyclooxygenase (COX)-2 in human kidney and to address localization of downstream targets for COX-derived prostanoids. Cortex and outer and inner medulla tissue were obtained from control kidneys (cancer specimens), kidneys with arterial stenosis, and kidneys of patients who received angiotensin II inhibition or acetylsalicylic acid. Ribonuclease protection assay and Western blot test revealed that COX-1 and -2 mRNA and protein were expressed in all regions of human kidney (mRNA ratio, cortex:outer medulla:inner medulla COX-1 1:3:20 and COX-2 1:1:3). In adult kidney, immunohistochemical labeling for COX-2 was associated with smooth muscle cells in pre- and postglomerular vessels and with endothelium, particularly in vasa recta and medullary capillaries. Western blot test confirmed COX-2 expression in renal artery. COX-2 had a similar localization in fetal kidney and was additionally observed in Henle's loop and macula densa. Human tissue arrays displayed COX-2 labeling of vascular smooth muscle in multiple extrarenal tissues. Vascular COX-2 expression was significantly increased in kidneys with arterial stenosis. COX-1 was colocalized with microsomal prostaglandin E(2) synthase (PGES) in collecting ducts, and PGES was also detected in macula densa cells. Vascular COX-2 was colocalized with prostaglandin E(2) EP4 receptors but not with EP2 receptors. Thus, renovascular COX-2 expression was a constitutive feature encountered in human kidneys at all ages, whereas COX-2 was seen in macula densa only in fetal kidney. Vascular COX-2 activity in human kidney and extrarenal tissues may support blood flow and affect vascular wall-blood interaction.     

Immunolocalization of multispecific organic anion transporters, OAT1, OAT2, and OAT3, in rat kidney

Recently, a family of multispecific organic anion transporters has been identified, and several isoforms have been reported. However, the physiologic and pharmacologic roles of each isoform, except OAT1, in the transepithelial transport of organic anions in the kidney remain to be elucidated. To address this issue, it is essential to determine the intrarenal distribution and membrane localization of each OAT isoform along the nephron. In this study, the intrarenal distributions of rOAT1, rOAT2, and rOAT3 were investigated by an immunofluorescence method that used frozen rat serial kidney sections. Confocal microscopic analysis showed that immunoreactivity for rOAT1 was detected exclusively in the proximal tubules (S1, S2, S3) in the cortex with basolateral membrane staining. rOAT2 was detected in the apical surface of the tubules in the medullary thick ascending limb of Henle's loop (MTAL) and cortical and medullary collecting ducts (CD). rOAT3 was localized in the basolateral digitation of the cell membrane in all the segments (S1, S2, and S3) of the proximal tubules, MTAL, cortical TAL, connecting tubules, and cortical and medullary CD. These results on the distribution of each OAT isoform will facilitate the understanding of the role of OATs in the renal processing of organic anions.     

The effect of deletion of cyclooxygenase-2, prostaglandin receptor EP2, or EP4 in bone marrow cells on osteoclasts induced by mouse mammary cancer cell lines

The inducible prostaglandin (PG) synthesis enzyme, cyclooxygenase-2 (COX-2), is involved in osteoclast (OC) formation in cocultures of mouse mammary cancer cell lines (MMT060562 or BALB/c-MC) and bone marrow cells through production of PGE(2). There are four PGE(2) receptors but only the EP2 and EP4 receptors are reported to be important for OC formation. We have investigated the role of COX-2, EP2 receptor, and EP4 receptor in marrow cells for osteoclastogenesis in cocultures of cancer cells and bone marrow cells. We cocultured cancer cell lines with bone marrow cells from COX-2 knockout (-/-), EP2 -/- or EP4 -/- mice compared to wild-type mice. In addition, an EP4 receptor antagonist (EP4 RA) was added in some cocultures. Disruption of COX-2 gene in bone marrow cells had no effect on PGE(2) production and OC formation in cocultures with MMT060562, while it abrogated PGE(2) production and OC formation in cocultures with BALB/c-MC. Disruption of the EP2 gene in bone marrow cells had no effect on OC formation in the cocultures, while disruption of the EP4 gene in bone marrow cells abrogated OC formation in the cocultures. Furthermore, EP4 RA suppressed OC formation and prevented the increase in receptor activator of nuclear factor kappaB ligand (RANKL) mRNA levels in the cocultures. We conclude that COX-2 in cancer cells is responsible for PGE(2) and OC production in cocultures with MMT060562, while COX-2 in bone marrow cells, not cancer cells, is responsible for PGE(2) and OC production in cocultures with BALB/c-MC, and EP4 receptors are essential for OC formation in both cocultures.     

Hypertonicity stimulates PGE2 signaling in the renal medulla by promoting EP3 and EP4 receptor expression

Hypertonicity in the renal medulla stimulates local cyclooxygenase 2 expression, leading to abundant PGE(2) production. Here we found that mRNA expression by the PGE(2)-activated G-protein-coupled receptors, EP3 and EP4 in the renal medulla was decreased by furosemide treatment, a procedure that reduces medullary hypertonicity. When HepG2 cells were cultured in hypertonic conditions by addition of salt or sorbitol, EP3 expression was induced. A specific EP3 agonist inhibited cAMP production, indicating receptor functionality, and this led to a substantial increase in cell survival in hypertonic media. Survival was independent of the SLC5A3 inositol transporter and aldose reductase expression, suggesting that EP3 promoted cell survival under hypertonic conditions independent of cellular organic osmolyte accumulation. Reduced cAMP production did not contribute to increased survival. EP4 expression was stimulated by hypertonicity in MDCK and HepG2 cells, which was associated with increased cAMP production in response to an EP4 agonist. Our study shows that local hypertonicity promotes PGE(2) signaling in the renal medulla by stimulating cognate receptor and cyclooxygenase 2 expression that likely regulates local hemodynamics and tubular transport.Kidney International (2009) 75, 278-284. doi:10.1038/ki.2008.498.     

Inhibition of interleukin-1beta-induced COX-2 and EP3 gene expression by sodium salicylate enhances pancreatic islet beta-cell function

Previous work has suggested that functional interrelationships may exist between inhibition of insulin secretion by interleukin (IL)-1beta and the endogenous synthesis of prostaglandin E(2) (PGE(2)) in the pancreatic islet. These studies were performed to ascertain the relative abundance of E prostaglandin (EP) receptor mRNAs in tissues that are major targets, or major degradative sites, of insulin; to identify which EP receptor type mediates PGE(2) inhibition of insulin secretion in pancreatic islets; and to examine possible sites of action through which sodium salicylate might affect IL-1beta/PGE(2) interactions. Real-time fluorescence-based RT-PCR indicated that EP3 is the most abundant EP receptor type in islets, liver, kidney, and epididymal fat. EP3 mRNA is the least, whereas EP2 mRNA is the most, abundant type in skeletal muscle. Misoprostol, an EP3 agonist, inhibited glucose-induced insulin secretion from islets, an event that was prevented by preincubation with pertussis toxin, by decreasing cAMP. Electromobility shift assays demonstrated that sodium salicylate inhibits IL-1beta-induced nuclear factor-kappaB (NF-kappaB) activation. Sodium salicylate also prevented IL-1beta from inducing EP3 and cyclooxygenase (COX)-2 gene expression in islets and thereby prevented IL-1beta from inhibiting glucose-induced insulin secretion. These findings indicate that the sites of action through which sodium salicylate inhibits these negative effects of IL-1beta on beta-cell function include activation of NF-kappaB as well as generation of PGE(2) by COX-2.     

SC-19220, antagonist of prostaglandin E2 receptor EP1, inhibits osteoclastogenesis by RANKL.

We examined the direct effect of SC-19220, an EP1 prostaglandin (PG) E2 receptor antagonist, on osteoclastogenesis induced by RANK/RANKL signaling in mouse cell cultures. We found that SC-19220 inhibited RANKL-induced osteoclastogenesis by suppression of the RANK/RANKL signaling pathway in osteoclast precursors. INTRODUCTION: Bone growth is accomplished by a dynamic equilibrium between formation by osteoblasts and resorption by osteoclasts, which are regulated by many systemic and local osteotropic factors that induce osteoclast formation from hematopoietic precursors through RANK/RANKL signaling. There are four subtypes of prostaglandin E (PGE) receptors, EP1, EP2, EP3, and EP4, and PGE2 facilitates bone resorption by a mechanism mediated by EP2/EP4. It is well known that SC-19220 is an EP1-specific antagonist. We previously found that SC-19220 inhibited osteoclastogenesis induced by osteotropic factors, including PGE2; however, the inhibitory mechanism is not clear. In this study, we investigated the inhibitory effects of SC-19220 on osteoclastogenesis induced by RANK/RANKL signaling in mouse cell cultures and analyzed the mechanism involved. MATERIALS AND METHODS: A bone marrow culture system and bone marrow macrophages were used to examine the effects of SC-19220 on PGE2-, 11-deoxy-PGE1-, and RANKL-induced osteoclastogenesis. We analyzed RANKL expression in osteoblasts induced by PGE2 using RT-PCR. We also examined the effects of SC-19220 on the macrophage-colony-stimulating factor (M-CSF) receptor (c-Fms) and RANK expression in osteoclast precursors as well as RANK/RANKL signaling using RT-PCR and Western blotting analyses. RESULTS AND CONCLUSION: SC-19220 dose-dependently inhibited osteoclast formation induced by PGE2, 11-deoxy-PGE1, and RANKL in the mouse culture system; however, it had no influence on RANKL expression in osteoblasts induced by PGE2. Furthermore, the expression of RANK and c-Fms in osteoclast precursors was decreased by SC-19220 at the mRNA and protein levels. In RANK signaling networks, SC-19220 inhibited c-Src and NFAT2 expression. Our findings indicated that SC-19220 inhibits RANKL-induced osteoclastogenesis through the suppression of RANK, c-Fms, c-Src, and NFAT2, suggesting that this EP1-specific antagonist inhibits osteoclast formation induced by RANKL from the early stage of osteoclastogenesis.     

Prostaglandin-E2 receptors in the rat kidney: biochemical characterization and localization

A radiohistochemical technique yielding data on the distribution and characteristics of PGE2-receptor binding in tissue sections is described. The binding of tritiated PGE2 (3H-PGE2) to slide-mounted tissue sections had all the characteristics associated with ligand-receptor interactions: it was saturable, of high affinity and displayed high specificity for PGE2 binding. From the binding curves a Hill coefficient of 1.1 was calculated which suggests the presence of a homogeneous receptor population. Pretreatment with indomethacin for four days resulted in a 66% increase in maximal binding capacity (Bmax) without any change in affinity. The distribution of receptor was mapped in rats with and without indomethacin pretreatment and compared to the distribution of Tamm-Horsfall glycoprotein, a specific marker for the thick ascending limb of Henle. In both groups the PGE2 receptor showed striking regional variation. In the untreated group the distribution of PGE2 receptors was similar to that of the thick ascending limb of Henle, with maximal density in the outer medullary zone. After indomethacin pretreatment, however, a striking increase in inner medullary binding was observed together with increased binding in the cortex. Thus, in accordance with the main action of PGE2 being regulation of renal water and sodium excretion, we found the highest receptor density in areas of the kidney dominated by the thick ascending limb of Henle and collecting tubules, and much less binding to glomeruli and cortical vessels. In order to investigate characteristics and distribution of PGE2 receptor binding, however, it was mandatory that endogenous prostanoid synthesis is blocked.     

Localization of inward rectifier potassium channel Kir7.1 in the basolateral membrane of distal nephron and collecting duct

Inward rectifier potassium channels (Kir) play an important role in the K(+) secretion from the kidney. Recently, a new subfamily of Kir, Kir7.1, has been cloned and shown to be present in the kidney as well as in the brain, choroid plexus, thyroid, and intestine. Its cellular and subcellular localization was examined along the renal tubule. Western blot from the kidney cortex showed a single band for Kir7.1 at 52 kD, which was also observed in microdissected segments from the thick ascending limb of Henle, distal convoluted tubule (DCT), connecting tubule, and cortical and medullary collecting ducts. Kir7.1 immunoreactivity was detected predominantly in the DCT, connecting tubule, and cortical collecting duct, with lesser expression in the thick ascending limb of Henle and in the medullary collecting duct. Kir7.1 was detected by electron microscopic immunocytochemistry on the basolateral membrane of the DCT and the principal cells of cortical collecting duct, but neither type A nor type B intercalated cells were stained. The message levels and immunoreactivity were decreased under low-K diet and reversed by low-K diet supplemented with 4% KCl. By the double-labeling immunogold method, both Kir7.1 and Na(+), K(+)-ATPase were independently located on the basolateral membrane. In conclusion, the novel Kir7.1 potassium channel is located predominantly in the basolateral membrane of the distal nephron and collecting duct where it could function together with Na(+), K(+)-ATPase and contribute to cell ion homeostasis and tubular K(+) secretion.     

Stimulation of prostaglandin EP2 receptors prevents NMDA-induced excitotoxicity

Prostaglandin E(2) (PGE(2)) plays an important role in inflammation and neurologic disorders. The neuromodulatory effects of PGE(2) are mediated through regulation of four G-protein-coupled receptors known as EP1, EP2, EP3, and EP4. The goal of the current study was to determine whether EP2 receptor activation protects neurons from acute NMDA-mediated excitotoxicity. To examine the effects of EP2 activation, mice were given an injection of the EP2 receptor-selective agonist butaprost (K (i) = 110 nM for EP2 receptor; K (i) > 10,000 for other prostaglandin receptors) in the cerebral ventricle and then an injection of NMDA in the right striatum. After 48 h, a significant reduction in NMDA-induced lesion volume was observed in groups pretreated with butaprost (1-300 nmol/L), with maximal protection at 100 nmol/L (p < 0.001). To determine if EP2-activated protection was specific to neurons, mouse neuronal cultures were treated with butaprost, and cell viability was analyzed after 24 h of NMDA excitotoxicity. The results showed that butaprost significantly increased neuron survival in a dose-dependent fashion. Furthermore, treatment of primary neurons with butaprost significantly increased cAMP levels (p < 0.001). Together, these data reveal that EP2 receptor stimulation mediates neuroprotection against NMDA excitotoxicity both in vivo and in vitro and that butaprost can limit acute brain damage. Development and testing of specific PGE(2) receptor mimetics could lead to a decrease in side effects associated with anti-inflammatory drugs and could help to fight acute and/or chronic neurologic disorders.