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BACKGROUND: Patients with diabetic nephropathy are at high risk for further progressive renal function loss. Treatments that decrease albuminuria have been linked with renal and cardiovascular protection. However, even when taking optimal treatment, residual renal and cardiovascular risk remains high which correlates with the magnitude of residual albuminuria. Use of vitamin D receptor activators, such as calcitriol and paricalcitol, is associated with improved sur- vival. A small study with paricalcitol showed reductions in albuminuria. The VITAL study tests the hypothesis whether paricalcitol persistently reduces albuminuria in diabetic subjects already receiving angiotensin-converting enzyme inhibitor (ACEI) and/or angiotensin receptor blocker (ARB) therapy. METHODS: Randomization in this double-blind trial is equal allocation to paricalcitol 1 micro/day, 2 microg/day, or placebo. Inclusion criteria include: a diagnosis of type 2 diabetes, urinary albumin/creatinine ratio (UACR) between 100-3,000 mg/g, estimated glomerular filtration rate (eGFR) between 15-90 ml/min/1.73 m(2), serum calcium <9.8 mg/dl, and parathyroid hormone (PTH) between 35-500 pg/ml. RESULTS: Baseline characteristics of the 281 subjects are: 69% men, mean age 64.9 +/- 10.4 years, eGFR 40.7 +/- 16.7 ml/min, median UACR (interquartile range) 612.3 mg/g (281-1,181 mg/g) and PTH 98.4 +/- 63.8 pg/ml. CONCLUSION: This trial will be the first clinical test of the hypothesis that paricalcitol possesses pleiotropic effects and can modulate albuminuria in the setting of ACEI and/or ARB therapy. Results will have important clinical implications and are expected in November 2009.

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AIMS: Cinacalcet lowers plasma parathyroid hormone (PTH) levels in patients with secondary hyperparathyroidism (sHPT), but the bone histologic response has not been described. This prospective, double-blind, placebo-controlled trial assessed the effects of cinacalcet on bone histology and serum markers of bone metabolism in dialysis patients with sHPT. METHODS: Patients with intact PTH (iPTH) > or = 300 pg/ml were randomly assigned 2:1 to receive cinacalcet or placebo with concurrent vitamin D and/or phosphate binder therapy. Cinacalcet (30 - 180 mg/day) was used to achieve iPTH levels < or = 200 pg/ml. Bone biopsies were performed before and after one year of treatment. RESULTS: Baseline and end-of-study data were available from 32 patients (19 cinacalcet, 13 placebo). Baseline bone turnover was elevated in 27, reduced in 3 and normal in 2 patients. Serum bone-specific alkaline phosphatase (BSAP) and N-telopeptide (NTx) were elevated. Cinacalcet treatment decreased PTH and diminished activation frequency, bone formation rate/bone surface, and fibrosis surface/bone surface. Adynamic bone was observed in three patients receiving cinacalcet; in two of these, PTH levels were persistently low (< 100 pg/ml). The histomorphometric parameter changes in bone corresponded to PTH, BSAP and NTx reductions. Bone mineralization parameters remained normal. CONCLUSIONS: Treatment with cinacalcet lowered PTH and reduced bone turnover and tissue fibrosis among most dialysis patients with biochemical evidence of sHPT.

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Treating hemodialysis patients to combat anemia corrects hemoglobin but exacerbates iron deficiency by utilizing iron stores. Patients needing iron should receive this by intravenous (i.v.) means. The Dialysis patients' Response to IV iron with Elevated ferritin (DRIVE) trial investigated the role of i.v. iron in anemic patients with high ferritin, low transferrin saturation, and adequate epoetin doses. We examined whether baseline iron and inflammation markers predict the response of hemoglobin to treatment. Patients (134) were randomized to no added iron or to i.v. ferric gluconate for eight consecutive hemodialysis sessions spanning 6 weeks with epoetin increased by 25% in both groups. The patients started with hemoglobin less than or equal to 11 g/dl, ferritin between 500 and 1200 ng/ml, and transferrin saturation of less than 25%. Significantly, patients with a reticulocyte hemoglobin content greater than or equal to 31.2 pg were over five times more likely to achieve a clinically significant increase in hemoglobin of greater than 2 g/dl. Lower reticulocyte hemoglobin contents did not preclude a response to i.v. iron. Significantly higher transferrin saturation or lower C-reactive protein but not ferritin or soluble transferrin receptor levels predicted a greater response; however their influence was not clinically significant in either group. We conclude that none of the studied markers is a good predictor of response to anemia treatment in this patient sub-population.

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BACKGROUND: Treatment of secondary hyperparathyroidism (SHPT) includes use of calcitriol (1,25D(3)) to suppress parathyroid hormone (PTH), but dosing of 1,25D(3) is limited by the development of hypercalcemia and a high calcium x phosphorus (Ca x P) product due to gut absorption of calcium and phosphorus as well as enhanced bone resorption. The vitamin D analog 19-Nor-1,25(OH)2-vitamin D2 (paricalcitol) and the prohormone 1alpha-OH-vitamin D2 (doxercalciferol) have been proposed as alternatives which may cause less hypercalcemia and elevated Ca x P, while still suppressing PTH. METHODS: We performed a prospective study to assess the acute bone mobilization effects of very high doses of paricalcitol and doxercalciferol. 13 hemodialysis patients received 160 mcg of paricalcitol and 120 mcg of doxercalciferol on 2 separate occasions in a research center while on a low calcium, low phosphorus diet, and sevelamer alone as a phosphorus binder. Changes in Ca, PO4, and PTH were measured over 36 h. RESULTS: Serum phosphorus rose faster, and peaked significantly higher at 36 h following doxercalciferol (2.12 +/- 0.11 mmol/l) than paricalcitol (1.85 +/- 0.07 mmol/l; p = 0.025). Ca x P product also rose more following doxercalciferol than paricalcitol, and peaked higher at 36 h (5.02 +/- 0.26 vs. 4.54 +/- 0.21 mmol/l; p = 0.061). In contrast, suppression of PTH at 36 h was comparable (63% after paricalcitol and 65% with doxercalciferol). CONCLUSION: Consistent with animal studies, paricalcitol provides profound PTH suppression, while stimulating bone resorption and/or intestinal absorption less than doxercalciferol, resulting in less elevation of serum phosphorus and Ca x P.

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BACKGROUND: Paricalcitol, a vitamin D analog, is commonly administered three times weekly to control secondary hyperparathyroidism in hemodialysis patients. Less frequent dosing would be more convenient, require less nursing time, and be an option in other dialysis modalities. No studies have examined the efficacy of once-weekly dosing of paricalcitol. METHODS: Chronic hemodialysis patients receiving a stable dose of paricalcitol three times weekly with intact PTH (iPTH) 100-500 ng/l were monitored during a two-week baseline, then were converted to a single mid-week paricalcitol dose equal to the previous cumulative weekly dose. Serum calcium and phosphorus were monitored weekly and iPTH levels determined during study Weeks 4 and 8. A single paricalcitol dose adjustment was made during study Week 5 based on iPTH to achieve a target value of 150-300 ng/l. Phosphate binders and calcium dialysate bath were kept constant during the study. RESULTS: In the 25 patients, mean iPTH was 295 +/- 107 ng/l at baseline, and not significantly different at Week 4 (307 +/- 111 ng/l) or Week 8 (285 +/- 98 ng/l). Paricalcitol dose increases mid-study were almost exclusively in patients with iPTH > 300 ng/l. Calcium, phosphorus, and calcium x phosphorus product were not significantly different on weekly therapy. (Only one patient developed a calcium > 2.55 mmol/l during the study.) CONCLUSION: Once-weekly dosing of paricalcitol is an effective option in treatment of secondary hyperparathyroidism. Less frequent dosing may better allocate nursing time and potentially benefit other patient populations with CKD and ESRD.

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Hepatitis B is easily spread via contact with infected blood. Hemodialysis patients and staff are particularly at risk for acquiring hepatitis B. Consequently, vaccination of hemodialysis patients and staff is strongly recommended. However, the vaccination rate among dialysis patients in this country remains below 50%. End-Stage Renal Disease (ESRD) Network 12 developed a quality improvement project directed at increasing patient vaccination by regular surveys, reports, and education of physicians, staff, and patients. Seventy-seven percent of facilities in the 4-state Network participated. Overall vaccination rate increased from 66.9% to 73.2% over 18 months (P <.05). The greatest improvement was seen among units with less than 60% of patients vaccinated initially, with mean facility vaccination rate increasing from 31.2 +/- 20.5% to 57.5 +/- 30.1% in the last available data period (P <.001). Only 3 of these 29 units failed to improve. The 90 units that had 60% to 97% vaccination rates initially improved significantly from 79.8 +/- 9.6% to 82.4 +/- 15% (P =.015). Three quarters of these units showed improvement. Only units with 100% vaccination deteriorated, but still maintained vaccination rates of 74.5 +/- 25.6%. An education-based quality improvement project can improve the hepatitis B vaccination rate of hemodialysis patients.

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BACKGROUND: Elevated plasma concentrations of leptin, a hormone thought to regulate body composition by influencing food intake/metabolic rate, are prevalent in renal failure patients. The mechanism for these increases is not known, but evidence suggests that simple accumulation due to decreased elimination is insufficient explanation. METHODS: We studied the incidence of hyperleptinaemia in 28 end-stage renal disease patients treated with continuous ambulatory peritoneal dialysis (CAPD), compared with body-mass-index-and sex-matched controls. Results were separated by gender because women have higher leptin concentrations than men. Excretion of leptin and other substances in dialysis fluid was also studied. RESULTS: Hyperleptinaemia was prevalent in women CAPD subjects, but not in men. Plasma leptin concentrations correlated strongly with the daily excretion of leptin in dialysis fluid. Clearance of leptin in dialysis fluid was greater in men than women CAPD subjects. Single regression analysis found that fasting insulin, glucose content of dialysis fluid, plasma albumin, C-reactive protein, erythropoietin dose, urinary creatinine clearance and plasma beta2-microglobulin were not determinants of plasma leptin concentrations. Stepwise forward multiple regression, examining the dependence of plasma leptin on body mass index, renal creatinine clearance, plasma albumin, daily dialysis fluid glucose load, daily leptin in dialysis fluid, erythropoietin dose and plasma C-reactive protein found only erythropoietin dose as a consistent negative predictor of plasma leptin concentrations. CONCLUSIONS: The results suggest that hyperleptinaemia of CAPD was due to predisposing loss of renal elimination capacity combined with increased production due to obesity (more prevalent in women subjects of this study) and potentially female gender.

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Leptin is a protein produced by fat cells and involved in body weight regulation. Plasma leptin is significantly higher in some hemodialysis (HD) patients than in normal controls. We examined the influence of dialyzer membrane biocompatibility and flux on elevated plasma leptin concentrations in hemodialysis patients. Employing a crossover design, leptin and tumor necrosis factor-alpha (TNF-alpha) levels were serially determined in eight chronic dialysis patients. Patients were dialyzed sequentially on low-flux cellulosic (TAF) dialyzers, low-flux (F8) polysulfone, high-flux (F80B) polysulfone, then low-flux polysulfone and cellulosic dialyzers again. Mean leptin concentrations were similar when low-flux polysulfone or cellulosic dialyzers were employed (141.9+/-24.2 microg/L versus 137.8+/-18.4 microg/L, respectively (P=NS). In contrast, leptin fell significantly on the high-flux polysulfone dialyzer (99.4+/-16.2 microg/L) compared with cellulosic (P < 0.005), and low-flux polysulfone dialyzers (P < 0.02). Leptin clearance by the high-flux polysulfone dialyzer was significantly higher than the low-flux dialyzers (50.4+/-21.5 v -9.6+/-10.3 mL/min; P=0.043), but did not account fully for the 30% decline in plasma leptin during the high-flux arm of the study. Concentrations of TNF-alpha were lower when high-flux polysulfone dialyzers were employed, but there was no correlation of individual TNF-alpha levels with leptin concentrations. High-flux dialysis lowers plasma leptin concentrations an average of 30%, but biocompatibility does not influence leptin levels. The decrease in plasma leptin on high-flux dialysis cannot be explained solely by enhanced clearance.

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BACKGROUND: Previous studies have reported that patients with end-stage renal disease (ESRD) have elevated plasma leptin concentrations, but the cause and significance of the elevations are unknown. We studied leptin concentrations in 29 adults undergoing renal transplantation, to determine if restoration of renal function reduced leptin concentrations in ESRD. METHODS: Leptin concentrations were measured by radioimmunoassay in plasma specimens collected within 1 week before transplant, 6 days post-transplant, and 60 days post-transplant. RESULTS: Mean plasma leptin concentrations were higher in both male and female ESRD patients compared with a control population of similar age and body mass index (BMI), but most of the disparity was due to a minority of patients with grossly elevated concentrations; the majority of ESRD patients had normal or near-normal leptin concentrations after accounting for their adiposity with BMI. Six days after successful renal transplantation, average plasma leptin concentrations decreased to control levels. The grossly elevated pretransplant concentrations in a minority of patients were greatly reduced in relation to BMI, and the reduction persisted to 60 days post-transplant. The decrease in creatinine with transplant did not correlate with the decrease in leptin. CONCLUSIONS: These results demonstrate that restoration of renal function in ESRD patients reduces hyperleptinaemia, which provides further evidence of a cause/effect relationship between impaired renal function and abnormal leptin metabolism.

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OBJECTIVE: To determine whether the increased plasma leptin levels reported in hemodialyzed patients is a feature of end-stage renal disease or an artifact of hemodialysis, we studied plasma levels in patients treated exclusively by continuous ambulatory peritoneal dialysis (CAPD). DESIGN: Prospective comparison of end points in CAPD patients and matched healthy subjects. SETTING: Tertiary care institutional dialysis center. PARTICIPANTS: Fifty-six healthy subjects, age 50.8 +/- 2.3 years, body mass index (BMI) 27.7 +/- 1.3 kg/m2, recruited through public announcement, and 36 patients with end-stage renal disease, age 51.0 +/- 2.4 yr, BMI 28.2 +/- 1.3 kg/m2, enrolled in a CAPD treatment program. INTERVENTION: Four exchanges of CAPD per day, using 2.0, 2.5, or 3.0 L of dialysate over a period of 1 - 96 months (median 22 mth). MAIN OUTCOME MEASURES: The primary outcome measure was plasma leptin concentration. Secondary measures included plasma glucose, insulin, C-peptide, and cortisol concentrations; and residual renal function and dialysis adequacy (Kt/V). RESULTS: Plasma leptin levels in CAPD patients were 27.1 - 490 ng/mL (women) and 1.3 - 355 ng/mL (men); the levels in healthy subjects were 2.0 - 84.7 ng/mL (women) and 1.8 - 55.4 ng/mL (men). The mean leptin levels were 5-fold higher among CAPD-treated men than control men (49.9 +/- 18.4 vs 9.8 +/- 2.5 ng/mL, p < 0.001) and 7.5-fold higher among CAPD-treated women than control women (220 +/- 28.1 vs 29.3 +/- 3.7 ng/mL, p < 0.0001). Female gender and BMI were the strongest predictors of hyperleptinemia in CAPD patients. CONCLUSION: These results indicate that hyperleptinemia is a feature of terminal renal failure, not an artifact of hemodialysis.

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PURPOSE: To determine the effectiveness of using Wallstents to treat subclavian or brachiocephalic venous obstruction after unsuccessful angioplasty, in patients undergoing hemodialysis. MATERIALS AND METHODS: Dialysis records, radiology reports, and procedural images of 20 hemodialysis patients who underwent Wallstent insertion into a subclavian (n = 11) or brachiocephalic (n = 9) vein were reviewed. Technical success and primary, assisted primary, and cumulative patency rates were calculated. RESULTS: Twenty-three Wallstents were inserted for stenosis (n = 18) or occlusion (n = 2). Technical success was 100%. Eight patients underwent 11 reinterventions to maintain patency of the Wallstent during the follow-up period. Patency rates of the Wallstent were (a) primary at 1 month, 3 months, 6 months, and 1 year: 90%, 67%, 42%, and 25%; (b) assisted primary at 3 months, 6 months, and 1 year: 88%, 62%, and 47%; and (c) cumulative at 3 months, 6 months, 1 year, and 2 years: 89%, 64%, 56%, and 22%. Considerable shortening of the stent occurred in five patients. One occurred immediately during the deployment procedure, but four were discovered weeks to months later. No other complications occurred. CONCLUSION: After suboptimal angioplasty, treatment of subclavian and brachiocephalic vein stenoses with a Wallstent can provide continued use of a hemodialysis access. Close clinical surveillance and multiple reinterventions are necessary to maintain Wallstent patency.

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Serial kinetic modeling is commonly used in hemodialysis to assess the adequacy of dialysis. A variety of problems lead to declining Kt/V in previously stable patients. These include noncompliance, vascular access recirculation, and dialyzer dysfunction. The purpose of this study was to find the relative frequencies of these problems in a group of patients undergoing routine hemodialysis. Simultaneous urea kinetic modeling and access recirculation were tested during 3 consecutive months. The baseline Kt/V was defined as the average of each patient's Kt/V values obtained during the previous 4 mo. A clinically important fall in Kt/V was defined as a decline of > or =0.2 if the baseline Kt/V was > or =1.2, or a decline of > or =0.1 if the baseline Kt/V was <1.2. Ninety-three of 375 (25%) sessions met the criteria for a significant decline in urea kinetic modeling. The baseline Kt/V in this group was 1.33 +/- 0.20 (mean +/- SEM) and declined to 1.02 +/- 0.18 in the abnormal month (P < 0.05). In 42% of instances with a decline of Kt/V, reduced blood processing due to a lower blood flow or shorter time than prescribed was responsible. Recirculation of >12% was found in 25% of sessions with a decrease in Kt/V. These patients most often had access dysfunction or reversed needles. The remaining one-third of patients with decreases in Kt/V had no problem identified, and subsequent monthly kinetic modeling results returned to baseline. These results suggest that analysis of falling urea kinetic modeling results should include a careful review of the dialysis record for reductions in prescribed time or blood flow rates followed by vascular access testing. If these evaluations are unrevealing, urea kinetic modeling results usually return to baseline in the next month.

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Leptin is a 16-kDa protein recently identified as the obese gene product involved in body weight regulation. Administration of recombinant leptin to ob/ob mice, which have a genetic defect in leptin production, reduces food intake and increases energy expenditure. Leptin is synthesized by fat cells, and in normal humans, plasma concentrations are proportional to adiposity. The physiological actions and the degradation pathways of leptin in humans are unknown. We investigated renal elimination of leptin by comparing plasma leptin concentrations in end-stage renal disease (ESRD) patients with normal controls. Our hypothesis was that if renal filtration is a significant route of elimination, the hormone would accumulate in ESRD patients. Mean plasma levels in 141 ESRD patients (26.8 +/- 5.7 and 38.3 +/- 5.6 micrograms/L for males and females, respectively) were significantly higher (P < 0.001) than mean values obtained in normal controls (11.9 +/- 3.1 and 21.2 +/- 3.0 micrograms/L for males and females, respectively). Leptin concentrations in ESRD patients correlated directly with body mass index (BMI; r = 0.77 for men and 0.78 for women). The rate of increase in leptin concentrations with BMI was significantly greater in ESRD patients (5.5 and 6.6 micrograms/L/U BMI for men and women, respectively) than in normal controls (1.4 and 2.6 micrograms/L/U for men and women, respectively). Pre- and postdialysis leptin levels in hemodialysis patients were similar. Western blot of plasma from ESRD patients with high leptin levels showed bands corresponding to the intact protein (16 kDa) with no lesser or greater molecular mass species observed. Leptin concentrations in patients with ESRD did not correlate with measures of residual renal function (serum creatinine, beta 2-microglobulin, PTH, or GH levels). Similarly, we found no correlation between leptin levels and the number of years patients had been on dialysis or with recent weight changes. We conclude that intact leptin is increased in ESRD patients, but does not appear to cause decreased weight. As leptin levels did not correlate with residual renal function, increased production may account for the high levels observed.

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BACKGROUND: Placement and maintenance of a well-functioning vascular access are essential for delivery of adequate hemodialysis. Newly placed polytetrafluoroethylene (PTFE) arteriovenous grafts require a period of wound healing and incorporation of fibrous tissue before use, a period typically lasting two to three weeks. An ideal PTFE graft would be one that can be used for vascular access immediately, obviating the need for temporary dialysis catheters. Recently an expanded PTFE (ePTFE) graft with a mesh cannulation segment (Diastat graft) has been proposed for early cannulation. STUDY DESIGN: This is a retrospective single-center study comparing ePTFE graft survival to contemporaneously placed standard wall PTFE (GORE-TEX) grafts. RESULTS: Forty-seven consecutive new or established patients receiving chronic hemodialysis had grafts (25 ePTFE, 22 standard PTFE) placed between November 1994 and July 1995. There were no significant differences between the groups in age, race, gender, incidence of diabetes mellitus, or peripheral vascular disease. By the end of the study, 21 of 25 ePTFE grafts had clotted, compared with 11 of the 22 patients receiving a standard PTFE graft. Median time to first clotting was 53 days for the ePTFE grafts and 164 days for the standard PTFE grafts (p < 0.0001). Nine patients with ePTFE grafts required a temporary catheter after their first clotting episode. CONCLUSIONS: The ePTFE grafts thrombosed at a significantly higher rate than standard wall PTFE grafts. Further experience with the Diastat graft might improve graft survival. However, early experience does not suggest that the avoidance of short-term temporary access outweighs the problem of high clotting rate, and its attendant morbidity.

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Platelet-derived growth factor (PDGF) is a disulphide-linked heterodimer of two polypeptide chains, the A and B chains, which are encoded by genes on separate chromosomes. The A-chain gene is transcribed in a number of transformed and non-transformed cell lines and is inducible by a wide variety of growth factors, cytokines and other mitogenic agonists. To localize DNA elements that mediate basal transcription in the promoter regulatory region of the A-chain gene, we have employed 5'-endpoint deletion mutagenesis and transient expression analysis in the renal epithelial cell line BSC-1 (African green monkey). Studies conducted in this cell line, which expresses high concentrations of PDGF A-chain mRNA, reveal a positive regulatory element (PRE) in a GC-rich stretch of the A-chain promoter between -82 and -40, relative to the transcription start site. Two discrete regions of the promoter were identified as negative regulatory elements (NREs), located between -1029 and -880 (NRE1) and between -1800 and -1029 (NRE2). The -1800 to -812 region, which contains both NREs, functions as a potent NRE when relocated in either orientation adjacent to the herpes simplex virus thymidine kinase promoter, reducing transcription activity by 60% in the positive orientation and 85% in the negative orientation. Comparison of BSC-1 cells and Saos-2 cells (human osteogenic sarcoma), which do not express significant quantities of PDGF A-chain mRNA or protein, indicates that basal transcription of the gene is determined by enhancer activity mediated by the GC-rich region rather than through de-repression of the upstream NREs. Electrophoretic gel mobility shift assays reveal a complex pattern of nuclear protein binding to the GC-rich PRE (-73 to -46). Competition studies conducted with mutant oligonucleotides that alternately disrupt consensus binding sites for Sp-1 or Egr-1 demonstrate a requirement for the presence of an Sp1-like core sequence (GGCGGG) but not Egr-1/Krox-24 [GCG(G/T)-GGGCG] for the formation of specific DNA-protein complexes. Our observations suggest that basal transcription of the A-chain gene in renal epithelial cells is achieved through active enhancement, mediated by a GC-rich PRE and nuclear proteins that bind to Sp-1-like consensus DNA sequences.

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Platelet-derived growth factor (PDGF) is a potent mitogen consisting of heterodimers of two distinct but homologous polypeptide chains, denoted A and B. PDGF-like homodimers of the A- and B-chains have been isolated, as well as two distinct receptor types (alpha and beta), which discriminate among the PDGF isoforms. The PDGF A- and B-chains are encoded by distinct genes located on human chromosomes 7 and 22, respectively. Although PDGF has been implicated as an important participant in development, tissue repair, and numerous pathologic states including tumorigenesis, atherosclerosis and inflammation, the mechanisms which determine the rate of its synthesis are only beginning to be understood. Basal expression of the PDGF A- and B-chain genes has been characterized in a number of cell types and is directed in part by elements in the respective proximal promoter-regulatory regions of the two genes. In addition, the first intron of PDGF-B has been shown to contain both positive and negative regulatory elements. Transcription of the PDGF subunit genes is inducible by a wide variety of mitogenic growth factors, cytokines and other agonists. These agents produce a rapid increase in steady-state concentrations of PDGF A- and B-chain mRNAs, peaking within 4-8 h of stimulation. The inductive effects of protein kinase C-activating phorbol 12-myristate 13-acetate (PMA), thrombin and transforming growth factor-beta (TGF-beta) are mediated through increases in the transcription rates of both genes. In addition, cAMP blocks the increases in transcription of the B-chain gene induced by thrombin and TGF-beta. Studies have demonstrated the importance of sequences immediately upstream of the B-chain transcription start site for induction in response to PMA-initiated megakaryocyte differentiation, an effect which is dependent on protein synthesis. However, cis-acting elements which mediate more rapid transcriptional induction seen in endothelial cells and astrocytes have yet to be identified in the proximal 5'-flanking sequences of either the A- or B-chain genes, suggesting that such events may be mediated by elements located outside of this region.

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The cytokines, interleukin-1 (IL-1) and tumor necrosis factor (TNF), potently induce prostaglandin formation in glomerular mesangial cells. Mechanisms by which these cytokines stimulate prostaglandin formation vary among cell types. We investigated whether alterations in phospholipase A2 (PLA2) or cyclooxygenase (COX) mass and activity contribute to the changes in mesangial cell prostaglandin production. These cytokines induced COX activity and mass in a time-dependent manner, which paralleled prostaglandin production. IL-1 increased COX mass approximately threefold by 24 h. TNF had a much smaller effect, although it appeared to be additive with IL-1. IL-1-induced COX mass was maintained at an increased level for at least 48 h. The glucocorticoid dexamethasone (DEX) virtually abolished prostaglandin production and blocked cytokine induction of COX activity and mass. DEX did not reduce COX activity or mass below the basal, serum-fed levels, however. By utilizing stable isotope methods, we could demonstrate that IL-1 increased free arachidonate levels, implying new PLA2 synthesis over a time course that was maximal at 6 h and was cycloheximide and actinomycin D sensitive. These data demonstrate that the cytokines IL-1 and TNF enhance synthesis of COX and PLA2, contributing to increased prostaglandin production. Cytokine-stimulated prostaglandin production ceases when cells are also treated with DEX, although control levels of COX activity and mass remain. This occurs because DEX inhibits the IL-1-induced enhanced arachidonate release.

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Prostaglandin production and cAMP formation are two signaling pathways identified for IL-1, though neither adequately account for the multitude of effects of IL-1. To investigate the role of tyrosine phosphorylation in IL-1 signaling, we used the tyrosine kinase inhibitor, genistein. At 10-30 micrograms/ml, genistein blocked IL-1 stimulated prostaglandin production and induction of prostaglandin endoperoxide synthase (PES) in glomerular mesangial cells maintained in 10% serum. Addition of genistein hours after IL-1 addition also halted further PGE2 synthesis. Genistein failed to block PES activity in vitro, indicating it was not acting as a PES inhibitor. Overall these data suggest that tyrosine phosphorylation may be a required event for IL-1 stimulation of PGE2 and PES activity, either directly as part of IL-1 signaling, or indirectly as part of a serum/PDGF competence effect on mesangial cells.

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