Renal Medullary Cells Research Articles

Aspirin Impairs Transport of Protective Osmolytes in Renal Inner Medullary Collecting Duct Cells

Non-steroidal anti-inflammatory drugs (NSAIDs) represent one of the most common classes of medications used worldwide for treatment of pain and inflammation, and their nephrotoxic effects have been well documented. This study is focused on aspirin action on the normal adaptive responses of cells in the hypertonic renal inner medulla, using a cultured cell model. Following adaptation to hypertonic conditions, further treatment of both MDCK and IMCD3 cells for 24h with 1.0 mM aspirin produced significant inhibition of the transport systems for the osmolytes betaine and taurine. Caspase 3 was not activated by this treatment, indicating that transport inhibition was not due to apoptotic events. Aceta- minophen and caffeine, common in analgesic mixtures, had a similar inhibitory effect. We conclude that interference with osmolyte accumulation by medullary cells may help to explain in part the nephrotoxicity due to long-term use of NSAIDs and analgesic formulations.

PGE2 potentiates tonicity-induced COX-2 expression in renal medullary cells in a positive feedback loop involving EP2-cAMP-PKA signaling

Cyooxygenase-2 (COX-2)-derived PGE2 is critical for the integrity and function of renal medullary cells during antidiuresis. The present study extended our previous finding that tonicity-induced COX-2 expression is further stimulated by the major COX-2 product PGE2 and investigated the underlying signaling pathways and the functional relevance of this phenomenon. Hyperosmolality stimulated COX-2 expression and activity in Madin-Darby canine kidney (MDCK) cells, a response that was further increased by PGE2-cAMP signaling, suggesting the existence of a positive feedback loop. This effect was diminished by AH-6809, an EP2 antagonist, and by the PKA inhibitor H-89, but not by AH-23848, an EP4 antagonist. The effect of PGE2 was mimicked by forskolin and dibutyryl-cAMP, suggesting that the stimulatory effect of PGE2 on COX-2 is mediated by a cAMP-PKA-dependent mechanism. Accordingly, cAMP-responsive element (CRE)-driven reporter activity paralleled the effects of PGE2, AH-6809, AH-23848, H-89, forskolin, and dibutyryl-cAMP on COX-2 expression. In addition, the stimulatory effect of PGE2 on tonicity-induced COX-2 expression was blunted in cells transfected with dominant-negative CRE binding (CREB) protein, as was the case in a COX-2 promoter reporter construct in which a putative CRE was deleted. Furthermore, PGE2 resulted in PKA-dependent phosphorylation of the pro-apoptotic protein Bad at Ser155, a mechanism that is known to inactivate Bad, which coincided with reduced caspase-3 activity during osmotic stress. Conversely, pharmacological interruption of the PGE2-EP2-cAMP-PKA pathway abolished Ser155 phosphorylation of Bad and blunted the protective effect of PGE2 on cell survival during osmotic stress. These observations indicate the existence of a positive feedback loop of PGE2 on COX-2 expression during osmotic stress, an effect that apparently is mediated by EP2-cAMP-PKA signaling, and that contributes to cell survival under hypertonic conditions.

Acute Hypertonicity Alters Aquaporin-2 Trafficking and Induces a MAPK-dependent Accumulation at the Plasma Membrane of Renal Epithelial Cells

The unique phenotype of renal medullary cells allows them to survive and functionally adapt to changes of interstitial osmolality/tonicity. We investigated the effects of acute hypertonic challenge on AQP2 (aquaporin-2) water channel trafficking. In the absence of vasopressin, hypertonicity alone induced rapid (<10 min) plasma membrane accumulation of AQP2 in rat kidney collecting duct principal cells in situ, and in several kidney epithelial lines. Confocal microscopy revealed that AQP2 also accumulated in the trans-Golgi network (TGN) following hypertonic challenge. AQP2 mutants that mimic the Ser(256)-phosphorylated and -nonphosphorylated state accumulated at the cell surface and TGN, respectively. Hypertonicity did not induce a change in cytosolic cAMP concentration, but inhibition of either calmodulin or cAMP-dependent protein kinase A activity blunted the hypertonicity-induced increase of AQP2 cell surface expression. Hypertonicity increased p38, ERK1/2, and JNK MAPK activity. Inhibiting MAPK activity abolished hypertonicity-induced accumulation of AQP2 at the cell surface but did not affect either vasopressin-dependent AQP2 trafficking or hypertonicity-induced AQP2 accumulation in the TGN. Finally, increased AQP2 cell surface expression induced by hypertonicity largely resulted from a reduction in endocytosis but not from an increase in exocytosis. These data indicate that acute hypertonicity profoundly alters AQP2 trafficking and that hypertonicity-induced AQP2 accumulation at the cell surface depends on MAP kinase activity. This may have important implications on adaptational processes governing transcellular water flux and/or cell survival under extreme conditions of hypertonicity.

Nitric oxide decreases expression of osmoprotective genes via direct inhibition of TonEBP transcriptional activity

During antidiuresis, renal medullary cells adapt to the hyperosmotic interstitial environment by increased expression of osmoprotective genes, which is driven by a common transcriptional activator, tonicity-responsive enhancer binding protein (TonEBP). Because nitric oxide (NO) is abundantly produced in the renal medulla, the present studies addressed the effect of NO on expression of osmoprotective genes and TonEBP activation in MDCK cells. Several structurally unrelated NO donors blunted tonicity-induced up-regulation of TonEBP target genes involved in intracellular accumulation of organic osmolytes. These effects were mediated by reduced transcriptional activity of TonEBP, as assessed by tonicity-responsive elements- and aldose reductase promoter-driven reporter constructs. Neither total TonEBP abundance nor nuclear translocation of TonEBP was affected by NO. Furthermore, 8-bromo-cGMP and peroxynitrite failed to reproduce the inhibitory effect of NO, indicating that NO acts directly on TonEBP rather than through classical NO signaling pathways. In support of this notion, electrophoretic mobility shift assays showed reduced binding of TonEBP to its target sequence in nuclear extracts prepared from MDCK cells treated with NO in vivo and in nuclear extracts exposed to NO in vitro. Furthermore, immunoprecipitation of S-nitrosylated proteins and the biotin-switch method identified TonEBP as a target for S-nitrosylation, which correlates with reduced DNA binding and transcriptional activity. These observations disclose a novel direct inhibitory effect of NO on TonEBP, a phenomenon that may be relevant for regulation of osmoprotective genes in the renal medulla.

Acute inhibition of the betaine transporter by ATP and adenosine in renal MDCK cells

Extracellular ATP interacts with purinergic P2 receptors to regulate a range of physiological responses, including downregulation of transport activity in the nephron. ATP is released from cells by mechanical stimuli such as cell volume changes, and autocrine signaling by extracellular ATP could occur in renal medullary cells during diuresis. This was tested in Madin-Darby canine kidney (MDCK) cells, a model used frequently to study P1 and P2 receptor activity. ATP was released within 1 min after transfer from 500 to 300 mosmol/kgH2O medium. A 30-min incubation with ATP produced dose-dependent inhibition (0.01-0.10 mM) of the renal betaine/GABA transporter (BGT1) with little effect on other osmolyte transporters. Inhibition was reproduced by specific agonists for P2X (alpha,beta-methylene-ATP) and P2Y (UTP) receptors. Adenosine, the final product of ATP hydrolysis, also inhibited BGT1 but not taurine transport. Inhibition by ATP and adenosine was blocked by pertussis toxin and A73122, suggesting involvement of inhibitory G protein and PLC in postreceptor signaling. Both ATP and adenosine (0.1 mM) produced rapid increases in intracellular Ca2+, due to the mobilization of intracellular Ca2+ stores and Ca2+ influx. Blocking these Ca2+ increases with BAPTA-AM also blocked the action of ATP and adenosine on BGT1 transport. Finally, immunohistochemical studies indicated that inhibition of BGT1 transport may be due to endocytic accumulation of BGT1 proteins from the plasma membrane. We conclude that ATP and adenosine, through stimulation of PLC and intracellular Ca2+, may be rapidly acting regulators of BGT1 transport especially in response to a fall in extracellular osmolarity.

Primary Culture and Characterization of Human Renal Inner Medullary Collecting Duct Epithelial Cells

Our understanding of physiological and pathophysiological events associated with inner medullary collecting duct epithelium is based on studies in cells isolated from mice and rats. We established primary cultures of hIMCD (human papillary collecting duct epithelial) cells. Normal papillary tissues were dissected from the surgical waste of consenting patients undergoing renal surgery. Tissues were digested enzymatically. Cells were maintained in Dulbecco's modified Eagle's medium supplemented with glucose and antibiotics. Cultures were treated with ethylenediaminetetraacetic acid and epithelial select medium was also used to obtain a pure epithelial culture. The hIMCD cells grew in a monolayer. Cells showed the expression of epithelial specific markers, including cytokeratin, the tight junction marker zonula occludens 1 and the cytoskeletal protein vimentin. They lacked expression of factor VIII, which is a glycoprotein synthesized by endothelial cells. To our knowledge we also noted for the first time uroplakin expression in collecting duct epithelial cells. This expression was maintained in primary culture. The hIMCD cells in culture were highly resistant to hypertonic solutions and they responded to hypertonicity by cyclooxygenase-2 over expression. Moreover, these cells also survived prolonged periods of hypoxia. To our knowledge this is the first report of successful culture and characterization of primary cultures of collecting duct epithelial cells from human renal papillae. These cells will serve as essential tools in helping us fill the gaps in our understanding of the events associated with the physiology and pathophysiology of human renal inner medullary collecting duct epithelium.

Hyperosmolality activates the integrated stress response in mouse medullary cells

The integrated stress response (ISR) is a eukaryotic translation initiation factor‐2 (eIF2a) phosphorylation‐dependent, stress inducible pathway activated by a variety of environmental stresses such as oxidative stress and endoplasmic reticulum stress. However IRS has not been characterized in osmotic stress. In normal conditions, renal medullary cells face high and various interstitial osmolality due to changing concentrations of NaCl and urea. To determine whether hyperosmolality causes IRS, mIMCD3 cells were subjected to hyperosmolality by adding extra NaCl or urea into culture medium. We found that phosphorylated eIF2a increased in the presence of 125, 150 or 200mM NaCl after 0.5, 1, 2 and 3 hours in a dose‐dependent manner. eIF2a phosphorylation also increased in response to 250, 300, 400mM urea at the same time points. High NaCl or urea activated an eIF2a kinase, general regulated non‐depressible‐2 (GCN2), in mIMCD3 cells. 7 day vasopressin infusion into mice raised the osmolality of the renal medulla and increased the mRNA expression of IRS pathway genes ATF3 and ATF4 (2.20 ± 0.37 fold and 1.62 ± 0.03, respectively, P&lt;0.05). We conclude that high concentrations of NaCl or urea activate IRS in renal medullary cells.

Disturbed water balance in Ku86−/‐ mice

High NaCl induces DNA breaks in cells in culture and in the kidney inner medulla in vivo. Ku86 binds to broken ends of damaged DNA and facilitates DNA repair. Ku86 deficiency compromises adaptation of cells to high NaCl, resulting in cellular hypertrophy, cell cycle arrest and chromosomal breakage (PNAS 102:10730,2005). We hypothesized that the renal function of Ku86−/‐ mice might be impaired because of increased sensitivity of their renal medullary cells to the high NaCl to which they are normally exposed as part of the urinary concentrating mechanism. To analyze the ability of Ku86−/‐ mice to maintain water balance, mice were subjected to different regimes of water consumption. During free access to water, Ku86−/‐ mice drink much more water leading to greater urine volume (2.45*±0.26 vs 1.11±0.09 ml/20g of body weight (BW)), and decreased urine osmolality compared to wild type mice. However, their urinary vasopressin excretion (U‐AVP) is significantly higher (271±76* vs 136±22 pg/24h per 20 g of BW). U‐AVP dramatically increases in Ku86−/‐ mice when water is limited to 3ml per 5g of gel food (599±74* vs 175±30 pg/24h) and they lose weight. Water restriction to 1.7ml/5g causes further weight loss in Ku86−/‐ mice. We conclude that Ku86−/‐ mice do not adapt normally to water restriction, possibly due to decreased responsiveness of renal collecting ducts to vasopressin. Supported by the Intramural Programs of NHLBI and NCI.

High NaCl Promotes Cellular Senescence

High extracellular NaCl was previously shown to increase the number of DNA breaks in mammalian cells in tissue culture, renal medullary cells in vivo, C. elegans, and marine invertebrates. It was also shown to increase reactive oxygen species in renal cells, resulting in oxidation of proteins and DNA. Cellular senescence is a common response to such damage. Therefore, in the present studies we looked for signs of senescence in cells exposed to high NaCl. We find that (1) The rate of proliferation of HeLa cells exposed to high NaCl decreases gradually to the point of arrest, and the cells display signs of senescence, including hypertrophy and increased auto fluorescence. (2) High NaCl accelerates the appearance of senescence in primary mouse embryonic fibroblasts, as measured by β-galactosidase activity (SA-β-gal). (3) High NaCl retards growth and markedly decreases the life span of C. elegans, accompanied by features of accelerated aging, such as decreased locomotion and increased number of SA-β-gal positive cells. (4) Mouse renal medullary cells, which are normally continuously exposed to high NaCl, express increased p16INK4 (another indicator of senescence) much earlier than do cells in the renal cortex, which has the same level of NaCl as peripheral blood. We conclude that high NaCl accelerates cellular senescence and aging, most likely secondary to the DNA breaks and oxidative damage that it causes.

Ectodomain shedding of pro-TGF-α is required for COX-2 induction and cell survival in renal medullary cells exposed to osmotic stress

In the renal medulla, cyclooxygenase (COX)-2 is induced by osmotic stress as present in this kidney region during antidiuresis. Increasing evidence suggests that EGF receptor (EGFR) signaling is involved in this process. The aim of the present study was to examine the mechanisms responsible for COX-2 expression and PGE(2) production during hypertonic conditions and to identify potential autocrine/paracrine EGFR ligands. Immunohistochemisty and Western blot analysis revealed abundant expression of the pro-EGFR ligand pro-transforming growth factor (TGF)-alpha in renal medullary cells in vivo and in cultured Madin-Darby canine kidney cells. In Madin-Darby canine kidney cells, hypertonicity rapidly increased TNF-alpha converting enzyme (TACE)-dependent ectodomain shedding of pro-TGF-alpha; phosphorylation of EGFR, p38, and ERK1/2; expression of COX-2; and production of PGE(2). Conversely, TACE inhibition prevented TGF-alpha release; EGFR, p38, and ERK1/2 activation; and COX-2 expression. Furthermore, cell survival was reduced substantially, a response that could be reversed by the addition of PGE(2). Simultaneous addition of recombinant TGF-alpha during TACE inhibition restored EGFR and MAPK phosphorylation, COX-2 expression, PGE(2) production, and cell survival during osmotic stress. These results indicate that hypertonicity induces TACE-mediated ectodomain shedding of pro-TGF-alpha, which subsequently activates COX-2 expression in an autocrine/paracrine fashion, via EGFR and MAPKs. We conclude that tonicity-induced TGF-alpha release is required for COX-2 expression, PGE(2) synthesis, and survival of renal medullary cells during osmotic stress.

Hypertonic sodium choloride and mannitol induces COX-2 via different signaling pathways in mouse cortical collecting duct M-1 cells

The kidney cortical collecting duct is an important site for the maintenance of sodium balance. Previous studies have shown that, in renal medullary cells, hypertonic stress induces expression of cyclooxygenase-2 (COX-2) via NF-κB activation, but little is known about COX-2 expression in response to hypertonicity in the cortical collecting duct. Therefore, we examined the mechanism of hypertonic induction of COX-2 in M-1 cells derived from mouse cortical collecting duct. Induction of COX-2 protein was detected within 6 h of treatment with hypertonic sodium chloride. The treatment also increased COX-2 mRNA accumulation in a cycloheximide-independent manner, suggesting that ongoing protein synthesis is not required for COX-2 induction. Using reporter plasmids containing 0.2-, 0.3-, and 1.5-kb fragments of the COX-2 promoter, we found that hypertonic induction of COX-2 was due to an increase in promoter activity. The COX-2-inductive effect of hypertonicity was inhibited by SB203580, indicating that the effect is mediated by p38 MAPK. Since p38 MAPK can activate NF-κB, we made point mutations in the NF-κB binding site within the COX-2 promoter. The mutations did not block the induction of COX-2 promoter activity by hypertonic sodium chloride, and hypertonic sodium chloride failed to activate NF-κB binding site-driven reporter gene constructs. In contrast, hypertonic mannitol activated NF-κB, indicating that hypertonic mannitol and hypertonic sodium chloride activate COX-2 by different mechanisms. Thus, induction of COX-2 expression in M-1 cells by hypertonic sodium chloride does not involve activation of NF-κB. Furthermore, the signal transduction pathways that respond to hypertonic stress vary for different osmolytes in cortical collecting duct cells.

Lack of Ku86 and water restriction both increase P16 INK4 expression in renal inner medullary cells in vivo.

High NaCl increases the number of DNA breaks in renal cells in culture and in the kidney inner medulla in vivo (Dmitrieva, PNAS, 2004). Ku86 is a DNA end binding protein that normally aligns the ends of broken DNA while it is being repaired. High NaCl disrupts chromatin, which leads to chromosomal aberrations. These are particularly pronounced in Ku86−/− cells (Dmitrieva, PNAS, 2005). P16INK4 is a member of INK4-class of cell cycle inhibitors. It is upregulated in cells whose DNA is damaged, preventing them from growing aberrantly. It is also upregulated in senescent cells and in cells that have exhausted their regenerative ability in the course of aging. In the present studies we show by immunohistochemical staining that the P16INK4 level in kidneys increases with age, and is highest in the inner medulla. Thus, in wild type mice the level of P16INK4 is higher in inner medulla than in the cortex, and is much greater at 12 months than at 3–4 months. Further, restricting water intake for two days increases renal p16INK4 level in wild type mice, and the greatest increase is in the inner medulla. In Ku86−/− mice, renal medullary p16INK4 is already high at 4 months, and the mice have damaged glomeruli. We speculate that the p16INK4level is elevated in renal medullas because of the normally high level of DNA breaks that occurs there and that deficiency of the DNA repair protein Ku86 exacerbates the response. The role and consequences of the high level of p16INK4remains to be established.

Urine Concentration and the Adaptation of Renal Medullary Cells to Hypertonicity

Hypertonicity (hypernatremia) of extracellular fluid causes water movement out of cells, while hypotonicity(hyponatremia) causes water movement into cells, resulting in cellular shrinkage or cellular swelling, respectively. In most part of the body, the osmolality of extracellular fluid is maintaine...

Neuropathy target esterase catalyzes osmoprotective renal synthesis of glycerophosphocholine in response to high NaCl

Glycerophosphocholine (GPC) is an osmoprotective compatible and counteracting organic osmolyte that accumulates in renal inner medullary cells in response to high NaCl and urea. We previously found that high NaCl increases GPC in renal [Madin-Darby canine kidney (MDCK)] cells. The GPC is derived from phosphatidylcholine, catalyzed by a phospholipase that was not identified at that time. Neuropathy target esterase (NTE) was recently shown to be a phospholipase B that catalyzes production of GPC from phosphatidylcholine. The purpose of the present study was to test whether NTE contributes to the high NaCl-induced increase of GPC synthesis in renal cells. We find that in mouse inner medullary collecting duct cells, high NaCl increases NTE mRNA within 8 h and NTE protein within 16 h. Diisopropyl fluorophosphate, which inhibits NTE esterase activity, reduces GPC accumulation, as does an siRNA that specifically reduces NTE protein abundance. The 20-h half-life of NTE mRNA is unaffected by high NaCl. TonEBP/OREBP is a transcription factor that is activated by high NaCl. Knockdown of TonEBP/OREBP by a specific siRNA inhibits the high NaCl-induced increase of NTE mRNA. Further, the lower renal inner medullary interstitial NaCl concentration that occurs chronically in ClCK1-/- mice and acutely in normal mice given furosemide is associated with lower NTE mRNA and protein. We conclude that high NaCl increases transcription of NTE, likely mediated by TonEBP/OREBP, and that the resultant increase of NTE expression contributes to increased production and accumulation of GPC in mammalian renal cells in tissue culture and in vivo.

Expression and actions of HIF prolyl-4-hydroxylase in the rat kidneys

Hypoxia inducible factor (HIF) prolyl-4-hydroxylase domain-containing proteins (PHDs) promote the degradation of HIF-1alpha. Because HIF-1alpha is highly expressed in the renal medulla and HIF-1alpha-targeted genes such as nitric oxide synthase, cyclooxygenase, and heme oxygenase are important in the regulation of renal medullary function, we hypothesized that PHD regulates HIF-1alpha levels in the renal medulla and, thereby, participates in the control of renal Na(+) excretion. Using real-time RT-PCR, Western blot, and immunohistochemical analyses, we have demonstrated that all three isoforms of PHD, PHD1, PHD2, and PHD3, are expressed in the kidneys and that PHD2 is the most abundant isoform. Regionally, all PHDs exhibited much higher levels in renal medulla than cortex. A furosemide-induced increase in renal medullary tissue Po(2) significantly decreased PHD levels in renal medulla, whereas hypoxia significantly increased mRNA levels of PHDs in cultured renal medullary interstitial cells, indicating that O(2) regulates PHDs. Functionally, the PHD inhibitor l-mimosine (l-Mim, 50 mg x kg(-1) x day(-1) i.p. for 2 wk) substantially upregulated HIF-1alpha expression in the kidneys, especially in the renal medulla, and remarkably enhanced (by >80%) the natriuretic response to renal perfusion pressure in Sprague-Dawley rats. Inhibition of HIF transcriptional activity by renal medullary transfection of HIF-1alpha decoy oligodeoxynucleotides attenuated l-Mim-induced enhancement of pressure natriuresis, which confirmed that HIF-1alpha mediated the effect of l-Mim. These results indicate that highly expressed PHDs in the renal medulla make an important contribution to the control of renal Na(+) excretion through regulation of HIF-1alpha and its targeted genes.

Survival in Hostile Environments: Strategies of Renal Medullary Cells

Cells in the renal medulla exist in a hostile milieu characterized by wide variations in extracellular solute concentrations, low oxygen tensions, and abundant reactive oxygen species. This article reviews the strategies adopted by these cells to allow them to survive and fulfill their functions under these extreme conditions.

Effects of expression of p53 and Gadd45 on osmotic tolerance of renal inner medullary cells

The response of renal inner medullary (IM) collecting duct cells (mIMCD3) to high NaCl involves increased expression of Gadd45 and p53, both of which have important effects on growth and survival of the cells. However, mIMCD3 cells, being immortalized by SV40, proliferate rapidly, which is known to sensitize cells to high NaCl, whereas IM cells in situ proliferate very slowly and survive much higher levels of NaCl. In the present studies, we have examined the importance of Gadd45 and p53 for survival of normal IM cells in their usual high-NaCl environment by using more slowly proliferating second-passage mouse inner medullary epithelial (p2mIME) cells and comparing cells from wild-type and gene knockout mice. Acutely elevating NaCl (and/or urea) reduces Gadd45a, but increases Gadd45b and Gadd45g mRNA, depending on the mix of NaCl and urea and the rate of increase of osmolality. Nevertheless, p2mIME cells from Gadd45b(-/-), Gadd45g(-/-), and Gadd45bg(-/-) mice survive elevation of NaCl (or urea) essentially the same as do wild-type cells. p53(-/-) Cells do not tolerate as high a concentration of NaCl (or urea) as p53(+/+) cells, but urinary concentrating ability of p53(-/-) mice is normal, as is the histology of inner medullas from p53(-/-) and Gadd45abg(-/-) mice. Thus although Gadd45 and p53 may play roles in osmotically stressed mIMCD3 cells, we do not find that their expression makes an important difference, either for Gadd45 in slower proliferating p2mIME cells or for Gadd45 or p53 in normal inner medullary epithelial cells in situ.

Renal PDE‐5 and the blunted natriuretic response to ANP in the gravid rat

Normal pregnancy is characterized by a large plasma volume expansion accompanied by sodium retention, which has been shown to be as a result of a blunted response to natriuretic stimuli, such as pressure natriuresis and atrial natriuretic peptide (ANP). PDE-5 protein expression has been shown to be increased in renal medullary cells during pregnancy. PDE-5 degrades cGMP to a non-active form, therefore we propose that increased PDE-5 availability causes in a fall in renal cGMP levels and blunts the natriuretic effect of ANP. We aimed to investigate the effect of intrarenal infusion of a selective PDE-5 inhibitor, sildenafil at 0.2μg/min, on the response to ANP in pregnancy. We performed acute experiments in anesthetized 16-day pregnant and virgin rats. ANP infusion caused a fall in BP in all groups and sildenafil induced a further reduction. Sodium excretion for the kidney receiving the intrarenal infusion is shown in Table 1. The sildenafil treatment resulted in restoring the increase in sodium excretion in the pregnant animals to levels equal to virgin animals. This data indicates that PDE-5 expression in the gravid rat kidney plays a role in the blunted natriuretic response to ANP during pregnancy and selective PDE-5 inhibition restores the natriuretic effect of ANP in the pregnant rat. Financial support for this work was provided by NIH, NICHD grant # R01 HD041571.

Role of Neuropathy Target Esterase (NTE) in high NaCl‐induced accumulation of glycerophosphocholine (GPC) by mouse Inner Medullary Collecting Duct 3 (mIMCD3) cells

GPC is an osmoprotective compatible and counteracting organic osmolyte that accumulates in renal inner medullary cells in response to high NaCl and urea. We previously found that high NaCl increases GPC in MDCK cells. The GPC is derived from phosphatidylcholine (PC), catalyzed by increased activity of a phospholipase that was not identified at that time. NTE was recently shown to be a phospholipase that catalyzes production of GPC from PC (Zaccheo et al, JBC 279: 24024, 2004). The purpose of the present studies was to test whether NTE is activated by high NaCl in mIMCD3 cells, resulting in increased GPC. We find that high NaCl increases NTE mRNA 3-fold within 8 hours, and NTE protein by 4-fold at 16 hours and 8-fold at 48 hours. GPC (measured with a sensitive chemiluminescent assay) increases 2.5-fold. Di-isopropyl FluoroPhosphate (DFP) inhibits NTE esterase activity. It reduces GPC accumulation by 30% at 300 mosmol/kg and by 40% at 500 mosmol/kg (NaCl added). Further, a specific siRNA, which reduces NTE protein abundance by 50% at both 300 and 500 mosmol/kg, reduces GPC to the same extent as DFP at both osmolalities. We conclude that high NaCl increases NTE expression and phospholipase B activity, contributing to increased production and accumulation of GPC in mIMCD3 cells.

Coordinate control of prostaglandin E2synthesis and uptake by hyperosmolarity in renal medullary interstitial cells

During water deprivation, prostaglandin E(2) (PGE(2)), formed by renal medullary interstitial cells (RMICs), feedback inhibits the actions of antidiuretic hormone. Interstitial PGE(2) concentrations represent the net of both PGE(2) synthesis by cyclooxygenase (COX) and PGE(2) uptake by carriers such as PGT. We used cultured RMICs to examine the effects of hyperosmolarity on both PG synthesis and PG uptake in the same RMIC. RMICs expressed endogenous PGT as assessed by mRNA and immunoblotting. RMICs rapidly took up [(3)H]PGE(2) to a level 5- to 10-fold above background and with a characteristic time-dependent "overshoot." Inhibitory constants (K(i)) for various PGs and PGT inhibitors were similar between RMICs and the cloned rat PGT. Increasing extracellular hyperosmolarity to the range of 335-485 mosM increased the net release of PGE(2) by RMICs, an effect that was concentration dependent, maximal by 24 h, reversible, and associated with increased expression of COX-2. Over the same time period, there was decreased cell-surface activity of PGT due to internalization of the transporter. With continued exposure to hyperosmolarity over 7-10 days, PGE(2) release remained elevated, COX-2 returned to baseline, and PGT-mediated uptake became markedly reduced. Our findings suggest that hyperosmolarity induces coordinated changes in COX-2-mediated PGE(2) synthesis and PGT-mediated PGE(2) uptake in RMICs.