Potential role of purinergic signaling in lithium-induced nephrogenic diabetes insipidus.

Lithium (Li)-induced nephrogenic diabetes insipidus (NDI) has been attributed to the increased production of renal prostaglandin (PG)E(2). Previously we reported that extracellular nucleotides (ATP/UTP), acting through P(2y2) receptor in rat medullary collecting duct (mCD), produce and release PGE(2). Hence we hypothesized that increased production of PGE(2) in Li-induced NDI may be mediated by enhanced purinergic signaling in the mCD. Sprague-Dawley rats were fed either control or Li-added diet for 14 or 21 days. Li feeding resulted in marked polyuria and polydipsia associated with a decrease in aquaporin (AQP)2 protein abundance in inner medulla ( approximately 20% of controls) and a twofold increase in urinary PGE(2). When acutely challenged ex vivo with adenosine 5'-O-(3-thiotriphosphate) (ATPgammaS), UTP, or ADP, mCD of Li-fed rats showed significantly higher increases (50-130% over control diet-fed rats) in PGE(2) production, indicating that more than one subtype of P(2y) receptor is involved. This was associated with a 3.4-fold increase in P(2y4), but not P(2y2), receptor mRNA expression in the inner medulla of Li-fed rats compared with control diet-fed rats. Confocal laser immunofluorescence microscopy revealed predominant localization of both P(2y2) and P(2y4) receptors in the mCD of control or Li diet-fed rats. Together, these data indicate that in Li-induced NDI 1) purinergic signaling in the mCD is sensitized with increased production of PGE(2) and 2) P(2y2) and/or P(2y4) receptors may be involved in the enhanced purinergic signaling. Our study also reveals the potential beneficial effects of P(2y) receptor antagonists in the treatment and/or prevention of Li-induced NDI.

Potential role of purinergic signaling in urinary concentration in inner medulla: insights from P2Y2 receptor gene knockout mice.

Osmotic reabsorption of water through aquaporin-2 (AQP2) in the inner medulla is largely dependent on the urea concentration gradients generated by urea transporter (UT) isoforms. Vasopressin (AVP) increases expression of both AQP2 and UT-A isoforms. Activation of the P2Y2 receptor (P2Y2-R) in the medullary collecting duct inhibits AVP-induced water flow. To gain further insights into the overarching effect of purinergic signaling on urinary concentration, we compared the protein abundances of AQP2 and UT-A isoforms between P2Y2-R knockout (KO) and wild-type (WT) mice under basal conditions and following AVP administration. Under basal conditions (a gel diet for 10 days), KO mice concentrated urine to a significantly higher degree, with 1.8-, 1.66-, and 1.29-fold higher protein abundances of AQP2, UT-A1, and UT-A2, respectively, compared with WT, despite comparable circulating AVP levels in both groups. Infusion of 1-desamino-8-d-arginine vasopressin (dDAVP; desmopressin; 1 ng/h sc) for 5 days resulted in 2.14-, 2.6-, and 2.22-fold higher protein abundances of AQP2, AQP3, and UT-A1, respectively, in the inner medullas of KO mice compared with WT mice. In response to acute (45 min) stimulation by AVP (0.2 unit/mouse sc), UT-A1 protein increased by 1.39- and 1.54-fold in WT and KO mice, respectively. These data suggest that genetic deletion of P2Y2-R results in increased abundances of key proteins involved in urinary concentration in the inner medulla, both under basal conditions and following AVP administration. Thus purinergic regulation may play a potential overarching role in balancing the effect of AVP on the urinary concentration mechanism.

Acute peritonitis in a C57BL/6 mouse model of peritoneal dialysis.

Studies using animal models of peritoneal dialysis (PD) have commonly induced acute peritonitis by intraperitoneal (IP) administration of lipopolysaccharide (LPS). We compared the effects of peritonitis induced by IP administration of either LPS or zymosan on inflammatory parameters [dialysate leukocyte counts and dialysate concentrations of prostaglandin E2 (PGE2) and vascular endothelial growth factor (VEGF)] and peritoneal transport of fluid, small solutes (glucose), and macromolecules (total protein) in a mouse model of PD. Eighteen hours after induction of peritonitis, mice were studied by injecting 2 mL of 4.25% dextrose-containing PD solution into the peritoneal cavity for a 2-hour dwell. Concentrations of glucose, total protein, PGE2, and VEGF were determined in the dialysate effluent. Acute peritonitis induced by IP administration of LPS induced changes in peritoneal transport similar to those observed during clinical PD, but without a significant increase in the dialysate leukocyte count. In contrast, acute peritonitis induced by IP administration of zymosan induced a large increase in dialysate leukocyte count, more substantial changes in peritoneal transport, and increases in dialysate PGE2 and VEGF concentrations. We conclude that acute peritonitis induced by IP administration of zymosan in the mouse may be a more relevant model for clinical PD, because it produces substantial changes in peritoneal transport and leukocyte migration into the peritoneal cavity.

Enhanced local production of vascular endothelial growth factor is not sufficient to increase peritoneal permeability to protein during acute peritonitis.

Dialysate concentrations of inflammatory mediators and growth factors, such as vascular endothelial growth factor (VEGF), are increased during acute peritonitis in peritoneal dialysis patients; however, it can be difficult to determine whether these high concentrations are caused by either increased peritoneal permeability or enhanced local production within peritoneal tissues. VEGF and total protein kinetics were first compared in a rabbit model during an 8-h dwell of dialysis solution containing 2.5% dextrose with (peritonitis) and without (control) the addition of 1-2 x 10(5) colony forming units (cfus) of Escherichia coli (series 1 experiments). Series 2 experiments determined whether intraperitoneal administration of indomethacin (75 microg/mL) altered the kinetics of VEGF and its local production during peritonitis. Series 1 experiments showed that peritonitis resulted in increased peritoneal permeability to total protein, enhanced appearance of VEGF in the dialysate, and increased tissue VEGF mRNA expression in the cecum and abdominal muscle tissues. Series 2 experiments showed that intraperitoneal administration of indomethacin during peritonitis blocked the increase in peritoneal permeability to total protein but had no effect on the appearance rate of VEGF in the dialysate. Intraperitoneal indomethacin decreased tissue VEGF mRNA expression in the cecum but not in the diaphragm or abdominal muscle tissues. It is concluded that the enhanced appearance of VEGF in peritoneal dialysate during peritonitis is largely from increased local production within peritoneal tissues. These observations also demonstrate that enhanced local production of VEGF is not sufficient to increase peritoneal permeability to total protein during peritonitis.

Effects of anaesthesia on fluid and solute transport in a C57BL6 mouse model of peritoneal dialysis.

BACKGROUND: Genetically modified mice show promise as animal models for studying the physiology and pathophysiology of the peritoneum during peritoneal dialysis (PD). Methods for evaluation of the functional characteristics of the mouse peritoneum have not been studied extensively, and the effects of anaesthesia on fluid and solute transport in mouse models of PD are unknown. METHODS: A single exchange of dialysis solution was performed in C57BL6 mice by injecting fluid into the peritoneal cavity using a 27-gauge needle and allowing fluid to dwell for 30, 60 or 120 min. Experiments evaluated the effect of ketamine (plus xylazine) anaesthesia on fluid and solute transport; these effects were examined in separate experiments using glucose and mannitol as the osmotic agent added to the injected dialysis solution. After euthanasia, blood was collected, the remaining dialysis solution was drained and their contents analysed for concentrations of the osmotic solute (glucose or mannitol), urea nitrogen (UN), sodium (Na) and a volume marker (fluorescein-labelled albumin) added to the initial, injected dialysis solution. Determined parameters included final volume of dialysis solution (drained plus residual fluid volume), dialysate concentration (D/D0) of glucose (or D/D0 mannitol), dialysate-to-plasma concentration ratio for (D/P) UN and D/P Na and the apparent dialysis solution volume by indicator dilution. Peritoneal permeability-area (PA) values or mass transfer-area coefficients were also calculated for the osmotic solutes. RESULTS: Final volumes of dialysis solution were higher when mice were anaesthetized with ketamine than in unanaesthetized mice, independent of whether glucose or mannitol was used as the osmotic agent. The increases in final volume were paralleled by higher dialysate concentrations (D/D0 values) and lower calculated PA values for both glucose and mannitol. When using either osmotic agent, anaesthesia also increased plasma glucose concentrations, suggesting that ketamine altered glucose metabolism. CONCLUSIONS: Ketamine anaesthesia in the mouse decreases PA values for glucose and mannitol when used as osmotic agents in PD solutions. The decrease in transperitoneal transport for these osmotic agents increases the final volume of fluid which can be obtained from the peritoneal cavity.

Dialyzer clearances and mass transfer-area coefficients for small solutes at low dialysate flow rates.

New daily hemodialysis therapies operate at low dialysate flow rates to minimize dialysate volume requirements; however, the dependence of dialyzer clearances and mass transfer-area coefficients for small solutes on dialysate flow rate under these conditions have not been studied extensively. We evaluated in vitro dialyzer clearances for urea and creatinine at dialysate flow rates of 40, 80, 120, 160, and 200 ml/min and ultrafiltration flow rates of 0, 1, and 2 l/h, using a dialyzer containing PUREMA membranes (NxStage Medical, Lawrence, MA). Clearances were measured directly across the dialyzer by perfusing bovine blood with added urea and creatinine single pass through the dialyzer at a nominal blood flow rate of 400 ml/min. Limited, additional studies were performed with the use of dialyzers containing PUREMA membranes at a blood flow rate of 200 ml/min and also with the use of other dialyzers containing polysulfone membranes (Optiflux 160NR, FMC-NA, Ogden, UT) and dialyzers containing Synphan membranes (NxStage Medical). For dialyzers containing PUREMA membranes, urea and creatinine clearances increased (p < 0.001) with increasing dialysate and ultrafiltration flow rates but were not different at blood flow rates of 200 and 400 ml/min. Dialysate saturation, defined as dialysate outlet concentration divided by blood water inlet concentration, for urea and creatinine was independent of blood and ultrafiltration flow rate but varied inversely (p < 0.001) with dialysate flow rate. Mass transfer-area coefficients for urea and creatinine were independent of blood and ultrafiltration flow rate but decreased (p < 0.001) with decreasing dialysate flow rate. Calculated mass transfer-area coefficients at low dialysate flow rates for all dialyzers tested were substantially lower than those reported by the manufacturers under conventional conditions. We conclude that dialyzers require specific characterization under relevant conditions if they are used in novel daily hemodialysis therapies at low dialysate flow rate.

Arterial and venous smooth-muscle cells differ in their responses to antiproliferative drugs.

Arteriovenous polytetrafluoroethylene (PTFE) grafts used for hemodialysis often fail as the result of myointimal hyperplasia with vascular smooth-muscle-cell (SMC) proliferation. The stenotic lesions occur primarily at the graft-vein anastomosis and less frequently at the graft-artery anastomosis. To explore the potentials of pharmacologic agents in preventing hemodialysis-graft stenosis, we first examined the susceptibility of venous and aortic SMCs to 3 antiproliferative drugs. Human aortic and saphenous-vein SMCs were cultured in a medium containing insulin, epidermal growth factor, fibroblast growth factor, and fetal bovine serum. Various concentrations of dipyridamole (0-100 microg/mL), paclitaxel (0-100 microg/mL), and tranilast (0-300 microg/mL) were added. After 72 hours, we subjected the cells to a mitochondrial enzymatic (methylthiazoletetrazolium; MTT) assay and a bromodeoxyuridine (BrdU)-incorporation assay as a means of assessing their proliferation. Dipyridamole, paclitaxel, and tranilast each inhibited the proliferation of aortic and venous SMCs in a dose-dependent manner ( P <.0001). Approximately 90% inhibition was achieved at dipyridamole concentrations of 75 microg/mL and greater in both MTT and BrdU assays; paclitaxel and tranilast were less effective. The venous SMCs were substantially more susceptible to inhibition by all 3 drugs than were the aortic SMCs in the MTT assay. The concentrations required to produce 50% inhibition (IC 50 ) in the venous cells were 5.8 microg/mL (11.5 micromol/L), 9.1 microg/mL (10.7 micromol/L), and 37.4 microg/mL (114.3 micromol/L), respectively, for dipyridamole, paclitaxel, and tranilast. These concentrations were approximately 4.2, 5.3, and 3.0 times lower, respectively, than the corresponding IC 50 values for the aortic cells. The differences in IC 50 between the aortic and venous cells for the 3 drugs were less pronounced in the BrdU assay. The results of this study suggest that strategies for the prevention of stenosis should take into account the fact that lesions at venous anastomoses of arteriovenous grafts may respond differently to drugs than do those at arterial anastomoses.

In vitro pharmacological inhibition of human vascular smooth muscle cell proliferation for the prevention of hemodialysis vascular access stenosis.

BACKGROUND: Vascular access for chronic hemodialysis often fails as a result of stenosis caused primarily by the proliferation of vascular smooth muscle cells (VSMC). Various drugs have been shown to inhibit the proliferation of VSMC under different conditions. METHODS: In this study, we compared the inhibitory effect of ten drugs on the proliferation of human aortic smooth muscle cells (SMC) in culture. Quiescent cells were cultured in the presence of growth factors, fetal bovine serum and incremental concentrations of the test drug. Cell proliferation was assessed by the MTT reduction assay. RESULTS: Aspirin, enalaprilat, heparin, hydroxyurea, indomethacin and tirofiban were ineffective. While dipyridamole, paclitaxel, tranilast and verapamil inhibited cell proliferation, the concentrations required were significantly higher than the clinical plasma levels achieved after systemic administration. CONCLUSION: Local delivery of these drugs to the target site may therefore be a more effective and appropriate strategy for the prevention of hemodialysis vascular access stenosis.

Hollow fiber shape alters solute clearances in high flux hemodialyzers.

The mass transfer properties of hemodialyzers containing hollow fiber membranes are known to be influenced by membrane chemical composition, surface area, and pore size; however, the effects of hollow fiber shape (or configuration) and packing density within the dialyzer housing have not been well characterized. We determined, both in vitro and ex vivo (clinical), solute clearances and mass transfer-area coefficients (KoA) for high flux dialyzers containing polysulfone hollow fibers of identical chemical composition but different shapes. Hemoflow F80A (1.8 m2 of membrane surface area) dialyzers contained hollow fibers with a conventional shape, but Optiflux F180A (1.8 m2), F200A (2.0 m2), and F200NR (2.0 m2) dialyzers contained hollow fibers with a wavy shape. Clearances and KoA values determined in vitro for urea and creatinine increased with increasing dialysate flow rate and were higher for Optiflux F180A and F200A dialyzers than for Hemoflow F80A dialyzers. In vitro clearances for lysozyme and myoglobin were also higher for Optiflux F180A and F200A dialyzers than for Hemoflow F80A dialyzers, suggesting that a wavy hollow fiber shape increases mass transfer by increasing effective membrane surface area, conceivably by altering dialysate flow patterns. Urea clearances and KoA values determined ex vivo were higher for Optiflux F200NR dialyzers than for Hemoflow F80A dialyzers, confirming that the in vitro results are applicable to clinical hemodialysis. These increases in mass transfer efficiency for dialyzers containing hollow fibers with a wavy shape are consistent with improved mass transfer within the dialysate compartment as evidenced by the manufacturer-reported dialysate pressure-flow relationships. We conclude that the mass transfer characteristics of high flux dialyzers can be altered by the shape of the hollow fibers.