Thiazolidinediones expand body fluid volume through PPARgamma stimulation of ENaC-mediated renal salt absorption.

Thiazolidinediones (TZDs) are widely used to treat type 2 diabetes mellitus; however, their use is complicated by systemic fluid retention. Along the nephron, the pharmacological target of TZDs, peroxisome proliferator-activated receptor-gamma (PPARgamma, encoded by Pparg), is most abundant in the collecting duct. Here we show that mice treated with TZDs experience early weight gain from increased total body water. Weight gain was blocked by the collecting duct-specific diuretic amiloride and was also prevented by deletion of Pparg from the collecting duct, using Pparg (flox/flox) mice. Deletion of collecting duct Pparg decreased renal Na(+) avidity and increased plasma aldosterone. Treating cultured collecting ducts with TZDs increased amiloride-sensitive Na(+) absorption and Scnn1g mRNA (encoding the epithelial Na(+) channel ENaCgamma) expression through a PPARgamma-dependent pathway. These studies identify Scnn1g as a PPARgamma target gene in the collecting duct. Activation of this pathway mediates fluid retention associated with TZDs, and suggests amiloride might provide a specific therapy.

Opposite effects of cyclooxygenase-1 and -2 activity on the pressor response to angiotensin II.

Therapeutic use of cyclooxygenase-inhibiting (COX-inhibiting) nonsteroidal antiinflammatory drugs (NSAIDs) is often complicated by renal side effects including hypertension and edema. The present studies were undertaken to elucidate the roles of COX1 and COX2 in regulating blood pressure and renal function. COX2 inhibitors or gene knockout dramatically augment the pressor effect of angiotensin II (Ang II). Unexpectedly, after a brief increase, the pressor effect of Ang II was abolished by COX1 deficiency (either inhibitor or knockout). Ang II infusion also reduced medullary blood flow in COX2-deficient but not in control or COX1-deficient animals, suggesting synthesis of COX2-dependent vasodilators in the renal medulla. Consistent with this, Ang II failed to stimulate renal medullary prostaglandin E(2) and prostaglandin I(2) production in COX2-deficient animals. Ang II infusion normally promotes natriuresis and diuresis, but COX2 deficiency blocked this effect. Thus, COX1 and COX2 exert opposite effects on systemic blood pressure and renal function. COX2 inhibitors reduce renal medullary blood flow, decrease urine flow, and enhance the pressor effect of Ang II. In contrast, the pressor effect of Ang II is blunted by COX1 inhibition. These results suggest that, rather than having similar cardiovascular effects, the activities of COX1 and COX2 are functionally antagonistic.

Luminal NaCl delivery regulates basolateral PGE2 release from macula densa cells.

Macula densa (MD) cells express COX-2 and COX-2-derived PGs appear to signal the release of renin from the renal juxtaglomerular apparatus, especially during volume depletion. However, the synthetic machinery and identity of the specific prostanoid released from intact MD cells remains uncertain. In the present studies, a novel biosensor tool was engineered to directly determine whether MD cells release PGE2 in response to low luminal NaCl concentration ([NaCl]L). HEK293 cells were transfected with the Ca2+-coupled E-prostanoid receptor EP1 (HEK/EP1) and loaded with fura-2. HEK/EP1 cells produced a significant elevation in intracellular [Ca2+] ([Ca2+]i) by 29.6 +/- 12.8 nM (n = 6) when positioned at the basolateral surface of isolated perfused MD cells and [NaCl]L was reduced from 150 mM to zero. HEK/EP1 [Ca2+]i responses were observed mainly in preparations from rabbits on a low-salt diet and were completely inhibited by either a selective COX-2 inhibitor or an EP1 antagonist, and also by 100 microM luminal furosemide. Also, 20-mM graduated reductions in [NaCl]L between 80 and 0 mM caused step-by-step increases in HEK/EP1 [Ca2+]i. Low-salt diet greatly increased the expression of both COX-2 and microsome-associated PGE synthase (mPGES) in the MD. These studies provide the first direct evidence that intact MD cells synthesize and release PGE2 during reduced luminal salt content and suggest that this response is important in the control of renin release and renal vascular resistance during salt deprivation.

Cloning and expression of the rabbit prostaglandin EP2 receptor.

BACKGROUND: Prostaglandin E2 (PGE2) has multiple physiologic roles mediated by G protein coupled receptors designated E-prostanoid, or "EP" receptors. Evidence supports an important role for the EP2 receptor in regulating fertility, vascular tone and renal function. RESULTS: The full-length rabbit EP2 receptor cDNA was cloned. The encoded polypeptide contains 361 amino acid residues with seven hydrophobic domains. COS-1 cells expressing the cloned rabbit EP2 exhibited specific [3H]PGE2 binding with a Kd of 19.1 +/- 1.7 nM. [3H]PGE2 was displaced by unlabeled ligands in the following order: PGE2>>PGD2=PGF2alpha=iloprost. Binding of [3H]PGE2 was also displaced by EP receptor subtype selective agonists with a rank order of affinity consistent with the EP2 receptor (butaprost>AH13205>misoprostol>sulprostone). Butaprost free acid produced a concentration-dependent increase in cAMP accumulation in rabbit EP2 transfected COS-1 cells with a half-maximal effective concentration of 480 nM. RNase protection assay revealed high expression in the ileum, spleen, and liver with lower expression in the kidney, lung, heart, uterus, adrenal gland and skeletal muscle. In situ hybridization localized EP2 mRNA to the uterine endometrium, but showed no distinct localization in the kidney. EP2 mRNA expression along the nephron was determined by RT-PCR and its expression was present in glomeruli, MCD, tDL and CCD. In cultured cells EP2 receptor was not detected in collecting ducts but was detected in renal interstitial cells and vascular smooth muscle cells. EP2 mRNA was also detected in arteries, veins, and preglomerular vessels of the kidney. CONCLUSION: EP2 expression pattern is consistent with the known functional roles for cAMP coupled PGE2 effects in reproductive and vascular tissues and renal interstitial cells. It remains uncertain whether it is also expressed in renal tubules.

Prostaglandin E2-EP4 receptor promotes endothelial cell migration via ERK activation and angiogenesis in vivo.

Prostaglandin E2 (PGE(2)), a major product of cyclooxygenase, exerts its functions by binding to four G protein-coupled receptors (EP1-4) and has been implicated in modulating angiogenesis. The present study examined the role of the EP4 receptor in regulating endothelial cell proliferation, migration, and tubulogenesis. Primary pulmonary microvascular endothelial cells were isolated from EP4(flox/flox) mice and were rendered null for the EP4 receptor with adenoCre virus. Whereas treatment with PGE(2) or the EP4 selective agonists PGE(1)-OH and ONO-AE1-329 induced migration, tubulogenesis, ERK activation and cAMP production in control adenovirus-transduced endothelial EP4(flox/flox) cells, no effects were seen in adenoCre-transduced EP4(flox/flox) cells. The EP4 agonist-induced endothelial cell migration was inhibited by ERK, but not PKA inhibitors, defining a functional link between PGE(2)-induced endothelial cell migration and EP4-mediated ERK signaling. Finally, PGE(2), as well as PGE(1)-OH and ONO-AE1-329, also promoted angiogenesis in an in vivo sponge assay providing evidence that the EP4 receptor mediates de novo vascularization in vivo.

Peroxisome proliferator-activated receptor delta activation promotes cell survival following hypertonic stress.

COX2-selective non-steroidal anti-inflammatory drugs (NSAIDs) cause selective apoptosis of renal medullary interstitial cells (RMIC) in vivo and reduce their ability to tolerate hypertonic stress in vitro. To determine the mechanism by which COX2 activity promotes RMIC viability, we examined the capacity of COX2-derived prostanoids to promote RMIC survival. Although RMICs synthesize prostaglandin E2 (PGE2) PGI2 > PGF2a > TxA2, only PGI2 enhanced RMIC viability following hypertonic stress. RMICs do not express the prostacyclin receptor, but they do express the prostacyclin responsive nuclear transcription factor peroxisome proliferator-activated receptor delta (PPARdelta). Hypertonic stress increased PGI2 synthesis 330% above base line and also activated a PPARdelta specific reporter (delta response element (DRE)) by 90% above base line. Conversely DRE activity was only inhibited by the COX2-selective inhibitor SC236 but not by a COX1-selective NSAID (SC560). Overexpression of PPARdelta using an adenovirus not only drove DRE activity but also prevented RMIC death due to COX2 inhibition. These studies are consistent with a model whereby hypertonicity activates COX2-derived prostaglandin production, which promotes RMIC viability through PPARdelta. Inhibition of PPARdelta activity may contribute to the renal papillary necrosis associated with analgesic and/or NSAID use.

Membrane-associated PGE synthase-1 (mPGES-1) is coexpressed with both COX-1 and COX-2 in the kidney.

BACKGROUND: Prostaglandin E2 (PGE2) plays an important role in many physiologic and pathophysiologic processes in the kidney. Multiple enzymes are involved in PGE2 biosynthesis, including phospholipases, cyclooxygenases (COX), and the PGE2 synthases (PGES). The present studies were aimed at determining the intrarenal localization of mPGES-1 and whether it is coexpressed with COX-1 or COX-2. METHODS: Rabbit mPGES-1 and COX-1 cDNAs were cloned using reverse transcription-polymerase chain reaction (RT-PCR) and screening a cDNA library. RNase protection assay and immunoblotting were used to examine mPGES-1 expression levels. In situ hybridization and immunostaining were used to determine the intrarenal localization of mPGES-1 and cyclooxygenases. RESULTS: Rabbit mPGES-1 shares high sequence similarity to the human homolog. Nuclease protection studies showed that the kidney expresses among the highest level of mPGES-1 of any rabbit tissue. In situ hybridization showed COX-1 and mPGES-1 mRNA was highly expressed in renal medullary collecting ducts (MCD), and to a lesser extent in cortical collecting ducts (CCD). Fainter mPGES-1 expression was also observed in macula densa (MD) and medullary interstitial cells (RMICs), where COX-2 is highly expressed. Double-labeling studies (immunostaining plus in situ hybridization) and immunohistochemistry of mouse tissues confirmed that mPGES-1 predominantly colocalizes with COX-1 in distal convoluted tubule (DCT), CCD, and MCD, and is coexpressed with COX-2 at lower levels in MD and RMICs. CONCLUSION: Together, these studies suggest mPGES-1 colocalizes with both COX-1 and COX-2 to mediate the biosynthesis of PGE2 in the kidney.