A permanent fish cell line (EPC) for genotoxicity testing of marine sediments with the comet assay.

Genotoxicity and cytotoxicity were evaluated in an in vitro system with a permanent cell line Epithelioma papulosum cyprini (EPC) derived from a skin tumour of carp (Cyprinus carpio L.). EPC cells were exposed to different concentrations of organic sediment extracts from the North Sea for 24h. After incubation the cells were analysed for viability and DNA strand breaks with the comet assay or single cell gel electrophoresis (SCGE). The results confirm the sensitivity of this assay. Out of 10 marine sediment samples from the North Sea, 9 showed a dose-dependent genotoxic effect. The EC(50) of sediment extracts ranged from 7 to 307 mg sediment dry weight/ml assay volume. Hepatic microsomal enzymes from dab (Limanda limanda L.) was proposed for enzymatic activation of benzo[a]pyrene (BAP) or sediment extracts, respectively. The suitability of this in vitro test system for assessing genotoxic and cytotoxic effects of marine sediment extracts on EPC cells could be demonstrated.

The poor accuracy of indirect measurements of cadaveric donor kidney weights.

Reports that examined the issue of whether transplantation of inadequate nephron mass may be a risk factor for long-term allograft failure yielded conflicting results. One of the more accurate methods of estimating glomerular mass is kidney weight. Most of the clinical studies used body surface area (BSA) or kidney length as estimates of kidney weight. To test the hypothesis that indirect measures of kidney weight are accurate estimates of kidney weight, we compared the kidney weight of 41 consecutive cadaveric kidneys to donor BSA, dimensions measured with calipers at the time of transplantation, and dimensions supplied by the Organ Procurement Agency (OPA). Linear regression analysis was used with kidney weight as the dependent variable and BSA, kidney length, or kidney volume as the independent variable. Kidney length measured with calipers was also compared to kidney length supplied by the OPA. Kidney weight had the best correlation with kidney volume and kidney length determined by caliper measurements (r = 0.640 and 0.646, respectively). The regression analysis showed that the correlation of kidney weight with BSA was 0.487. The correlation of OPA-provided kidney length with kidney weights was poor (r = 0.410). The linear regression of caliper-measured kidney length versus OPA length yielded a slope of 0.360, instead of an ideal slope of 1. The assumption has been made that kidney weight or a surrogate of kidney weight has an excellent correlation with nephron mass. Some of the variability in studies that attempted to examine the effect of transplanted nephron mass on allograft outcome may be due to inaccurate estimates used for kidney weight. Our data suggest that surrogate measurements of kidney weight may not be accurate. We recommend that measured kidney weight should be used in studies examining the effect of donor renal mass on allograft outcomes.

Risk factors for peritonitis in long-term peritoneal dialysis: the Network 9 peritonitis and catheter survival studies. Academic Subcommittee of the Steering Committee of the Network 9 Peritonitis and Catheter Survival Studies.

To determine factors involved in peritoneal dialysis-associated peritonitis and catheter loss, all point prevalent peritoneal dialysis patients in Health Care Finance Administration (HCFA) end-stage renal disease (ESRD) Network 9 were followed throughout 1991 for peritonitis events and throughout 1991 to 1992 for catheter survival. Data were collected by questionnaires compiled by the dialysis facility and validated by network staff. Peritonitis was reported 1,168 times in 729 of the 1,930 patients. By gamma-Poisson regression, a significantly increased risk for peritonitis was observed for patients with previous peritonitis, black race, and those dialyzing with standard connectors or cyclers compared with disconnect systems. Decreased risks were observed for patients with longer ESRD experience and when prophylactic antibiotics were administered before catheter insertion. Postinsertion leakage, diabetes, visual problems, previous or current immunosuppression, and physical activity were not risk factors. Infection of any kind caused the removal of 68% of the 414 catheters lost. Patients with downward-directed tunnels were less likely to experience concomitant exit site/tunnel infections associated with peritonitis. Peritonitis episodes with Staphylococcus epidermidis-like organisms were more likely to resolve with a single course of antibiotics. Perhaps because of their higher infection rate, blacks were more likely than whites to use a disconnect system. In general, the outcome of peritonitis in blacks was similar to that in whites, except that blacks were less likely to be hospitalized and were less likely to die.

Pseudomonas peritonitis in peritoneal dialysis patients: the Network #9 Peritonitis Study.

To determine risk factors for the development of Pseudomonas peritonitis (PsP) and outcomes of PsP, the authors compared peritoneal dialysis patients who developed PsP with peritoneal dialysis patients who developed non-Pseudomonas bacterial peritonitis (non-PsP). The authors also sought to determine if there were differences in patients who had resolution of PsP compared with those patients whose PsP did not resolve. The data were derived from the prospective Tristate Renal Network Peritonitis and Catheter Survival Study. Resolution in this study was defined as clearing of peritoneal dialysate on visual inspection, with up to three courses of antibiotic therapy allowed. Catheter removal, switch to hemodialysis, or death were outcomes that were considered separately from resolution because of the study design. There were 31 cases of PsP in 28 patients and 886 cases of non-PsP identified in 667 adult patients. There were no differences in race, gender, age, or incidence of diabetes between the groups. The PsP group had a 25% incidence of previous exposure to immunosuppressive agents, whereas it was 10.6% in the non-PsP group (P = 0.028). PsP infections were more frequently associated with concomitant exit and tunnel infections, higher hospitalization rates, increased incidence of catheter loss, switch to hemodialysis, and a worse rate of resolution when compared with non-PsP (all, P < 0.05). Logistic regression could not identify patients at increased risk of PsP. PsP resolved with antibiotic therapy only in 10 of 31 episodes.(ABSTRACT TRUNCATED AT 250 WORDS)

An improved glomerular filtration rate in cardiac transplant recipients with once-a-day cyclosporine dosing.

We tested the hypothesis that there would be a difference in the unwanted side effects of cyclosporine (CsA) when heart transplant patients received CsA once a day versus half the dose twice a day. Eight stable cardiac transplant patients (> 6 months posttransplant) were administered their dose of CsA either as a once-a-day dose or half the dose b.i.d. for 21 days in a random fashion. After 21 days the patients were crossed over to the other regimen. Patients underwent inulin and PAH clearances at CsA trough on each arm of the study. Each patient collected several 24-hr urines for determination of creatinine clearance, and had ambulatory blood pressure monitoring done during each arm of the study. Serum chemistries and lipid profiles were performed at the end of each arm of the study. The CsA dose was 1.9-7.2 mg/kg/day. All patients were hypertensive and on calcium channel antagonists. Once-a-day CsA dosing resulted in a 29% decrease in trough CsA levels. A significant increase in glomerular filtration rate, as estimated by the clearance of inulin, (65.16 +/- 24.4 q. day vs. 54.62 +/- 19.0 b.i.d. (ml/min) P < .02) and a significant increase in renal plasma flow, as estimated by the clearance of PAH, occurred with once-a-day dosing when compared with b.i.d. dosing (P = .02). Creatinine clearances were not different between the 2 arms of the study and significantly overestimated glomerular filtration rates (P = .01). CsA dosing b.i.d. resulted in significantly higher nocturnal blood pressures (91.2 +/- 8.3 b.i.d. vs. 86.4 +/- 8.1 q. day, mmHg, P = .015) when compared with once-a-day dosing. A significant increase in LDL cholesterol and a significant decrease in HDL cholesterol were noted during the b.i.d. dosing arm when compared with q. day CsA dosing. We conclude that in stable heart transplant patients once-a-day CsA dosing results in better GFR and renal plasma flow, a lower nocturnal blood pressure, and an improved lipid profile when compared with dosing CsA twice a day.

Effect of erythropoietin on renal excretion of a sodium load.

To determine whether erythropoietin alters the renal excretion of a sodium load in humans, we administered either erythropoietin (150 units/kg) or vehicle intravenously in a randomized crossover design to six normal white men on day 4 of a controlled sodium diet. After erythropoietin administration, the subjects were given 2 L normal saline solution intravenously over 4 hours. All urine was collected for 4 days after drug administration. Serum sodium and creatinine levels and blood pressure were determined 0, 4, 8, and 24 hours after drug administration. Peripheral renin activity and aldosterone levels were determined 0, 4, and 8 hours after drug administration. Erythropoietin significantly decreased total sodium excretion during the 4 days after drug administration (erythropoietin = 784 +/- 46 mEq/4 days versus control = 840 +/- 41 mEq/4 days; p < 0.001). Erythropoietin did not alter creatinine clearance, blood pressure, or the changes in plasma renin activity and aldosterone caused by the saline load. We conclude that erythropoietin decreases urine sodium excretion after a sodium load in normal human subjects without altering glomerular filtration rate, blood pressure, or plasma renin activity.

Culture-negative CAPD peritonitis: the Network 9 Study.

A portion of peritonitis episodes are reported as culture-negative or as initial no growth peritonitis (INGP). To determine if demographics, symptoms, signs, treatment, and outcome were different in INGP when compared to culture-positive peritonitis (Pos), we examined data from the Network 9 Peritonitis and Catheter Survival Study. Only peritonitis episodes occurring in adults with peritoneal dialysis (PD) fluid WBC counts greater than 100 were included in the analysis. INGP accounted for 14% of the episodes of peritonitis. Organisms grew out of 13 of the 37 patients in INGP that were recultured: 3 fungal, 5 gram-negative, and 5 gram-positive isolates. A difference in culture methodology for the two groups could not be detected. There was no difference in gender, race, incidence of diabetes, previous peritonitis, or exit-site infections between the two groups. INGP had a greater percentage of patients over age 70 (23.3% vs 14.7%, p < 0.05), and a larger percentage of INGP patients placed additives in their dialysate (55% vs 43.6%, p < 0.05). There was no difference in symptoms or signs between the two groups. The INGP group had half the catheter removal rate (9/103 vs 110/630 for Pos, p < 0.05), otherwise, there was no difference in the rate of hospitalization, death, or switch to hemodialysis. There was no difference in types of drugs used or method of drug administration between the two groups. A lower percentage of INGP patients were treated for 6 days or less and a higher percentage received 7-10 days of intraperitoneal (IP) therapy when compared to Pos.(ABSTRACT TRUNCATED AT 250 WORDS)

Infrequent dosing of subcutaneous erythropoietin for the treatment of anemia in patients on CAPD.

Erythropoietin (EPO) given subcutaneously (SC) once per week has been successful in the treatment of anemia in continuous ambulatory peritoneal dialysis (CAPD) patients. We have identified a population of CAPD patients that requires EPO administration once per week or less often. To determine if specific variables could be identified that would predict which CAPD patients would require infrequent EPO dosing, we reviewed the charts of all our CAPD patients who were receiving EPO as of 1 June 1992. Patients had to have been on CAPD for 3 months and EPO for 3 months to be considered for analysis. We identified 12 patients who required EPO once per week or less frequently (infrequent EPO) and 9 patients who required EPO more than once per week (frequent EPO). Parameters that were analyzed included age, gender, race, time on CAPD, history of gastrointestinal bleeding, exit-site infection or peritonitis in the last 60 days, diabetes, amount of dialysate instilled per day, and the number of exchanges per day. Laboratory data that were analyzed included hemoglobin, hematocrit, serum iron, total iron-binding capacity, ferritin, blood urea nitrogen (BUN), creatinine, BUN/creatinine ratio, albumin, total protein, parathyroid hormone, and aluminum. Categorical data were analyzed via chi-square, and numerical data were analyzed via the t-test. The infrequent EPO group required only 35% as much EPO as the frequent group to maintain hemoglobin and hematocrit, which were significantly greater. The only parameter that was different between the two groups was age (infrequent EPO 42 +/- 13.2 vs frequent EPO 55.8 +/- 11.9 years, p < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of calcium channel antagonists on renal function in hypertensive heart transplant recipients.

Orthotopic heart transplant recipients treated with immunosuppressive regimens based on cyclosporine have a high incidence of hypertension. Cyclosporine-induced nephrotoxicity characterized by afferent glomerular arteriolar vasoconstriction also develops in these patients. Calcium channel antagonists produce afferent glomerular arteriolar vasodilation. Angiotensin-converting enzyme inhibitors (ACEI) dilate the efferent arteriole and have been suggested to decrease glomerular filtration rate in subjects taking cyclosporine. To test the hypothesis that calcium channel antagonists would improve glomerular filtration rate in heart transplant patients receiving ACEI treatment, we reviewed the charts of our patients whose treatment for hypertension had been changed from an ACEI to a calcium channel antagonist. A change in renal function was assessed by the average of serum creatinine level, blood urea nitrogen, and creatinine clearance within 3 months before and after the change from ACEI to calcium channel antagonist. Blood pressure was assessed on two different occasions before and after conversion to calcium channel antagonist. The data were analyzed by a paired Student t test. Serum blood urea nitrogen and creatinine levels decreased significantly when patients were treated with calcium channel antagonists (p < 0.05). Creatinine clearance increased in all patients when the treatment was converted to a calcium channel antagonist (CCA) (ACEI = 56.4 +/- 19.3 ml/min versus CCA = 71.06 +/- 23.77, N = 9; p < 0.005). A 5-mm Hg decrease occurred in mean arterial pressure when treatment was changed from ACEI to calcium channel antagonists.(ABSTRACT TRUNCATED AT 250 WORDS)

Protection of glomerular filtration rate by the thromboxane receptor antagonist L655,240 during low dose cyclosporine administration.

Cyclosporin A (CsA) alters the production of prostaglandins (PG) by the kidney. CsA causes an increase in renal vascular resistance, a decrease in renal blood flow, a decrease in glomerular filtration rate (GFR), and increases the renal production of the vasoconstrictor thromboxane. Recently, low dose CsA has been utilized in the treatment of refractory autoimmune diseases. To determine if low dose CsA administration could produce renal hemodynamic alterations and to determine if the thromboxane receptor antagonist L655,240 could prevent these alterations, we administered groups of rats either CsA, 5 mg/kg, subcutaneously and the L655,240 vehicle NaHCO3 (CsA-NaHCO3), or CsA and L655,240 (CsA-L655,240), or CsA vehicle and L655,240. The rats were administered the drugs for 7 days and then subjected to inulin and PAH clearances or kidneys were harvested for prostaglandin production studies. CsA significantly depressed GFR and renal plasma flow when compared to the L655,240 treated groups. There was no difference in inulin or PAH clearance between the CsA-L655,240 and CsA vehicle L655,240 groups. Glomerular prostaglandin production including thromboxane was depressed by CsA administration. No histologic alterations were noted in the glomeruli or the medullary portions of the kidney. We conclude that administration of low dose CsA, 5 mg/kg, for 7 days results in a decrease in renal blood flow and GFR without histologic alterations. Administration of the thromboxane receptor antagonist L655,240 prevents the renal hemodynamic alterations induced by CsA in this rat model.

The effect of cyclosporine on agonist-stimulated glomerular and mesangial cell vasodilatory prostaglandin production.

Cyclosporine A administration produces an increase in renal vascular resistance and a decrease in glomerular filtration rate in both human and animal models. CsA usage in humans has also been shown to alter the ability of the kidney to adapt to alterations in renal hemodynamics. CsA alters the production of prostaglandins by isolated rat glomeruli. Normally, vasoconstrictive agents stimulate the production of vasodilatory glomerular and mesangial cell PG. To determine if CsA alters glomerular and mesangial cell (MC) vasodilatory PG production in response to vasoconstrictive agents, we administered CsA, 20 mg/kg, or vehicle to rats for 7 days, or incubated mesangial cells with CsA 1 mcg/ml for 24 hr. Ex vivo glomerular PGE2 and 6-keto-PGF1a production was determined in the presence or absence of angiotensin II 10(-6) M and norepinephrine 10(-5)M. CsA administration decreased glomerular production of both eicosanoids in the basal and stimulated state. Incubation of MC with CsA markedly suppressed PGE2 and 6-keto-PGF1a production in response to stimulation with 200 nM angiotensin II. To determine if CsA inhibits angiotensin II-stimulated PG production prior to protein kinase C, we incubated glomeruli and MC with the diacylglycerol mimetic OAG. CsA depressed OAG-stimulated glomerular and MC PGE2 and 6-keto-PGF1a production. Conversely, CsA stimulated the production of PGE2 by renal medullary slices. We conclude that CsA blunts the vasoconstrictor-induced increase in glomerular and mesangial cell vasodilatory PG production, thereby removing a compensatory mechanism that maintains GFR in states of vasoconstrictor excess.

Urinary excretion and renal production of prostaglandins E2, F2 alpha, and thromboxane B2 in experimental diabetes mellitus.

To delineate the urinary excretion of prostaglandins E2 (PGE), F2 alpha (PGF), and thromboxane B2 (TxB) in diabetic rats, we treated male Sprague-Dawley rats with streptozocin 60 mg/kg or with vehicle. Plasma glucose and creatinine concentration and 24-hour urine collections for determination of TxB, PGE, PGF, creatinine, and protein excretion were measured at 2, 8, and 14 weeks after streptozocin. Creatinine clearance was significantly decreased in diabetic rats at 2 and 8 weeks, whereas the urinary protein excretion was three to five times that of control animals at all times. The 24-hour excretion of TxB was elevated twofold to threefold in diabetic rats, whereas PGE and PGF excretion were both significantly decreased. This alteration in excretion was not caused by increased urine flow rate, inasmuch as mannitol-induced osmotic diuresis caused an increase in PGE and PGF excretion. Eight weeks after streptozocin, a group of diabetic and control rats were sacrificed, and the production of PGE, PGF, and TxB by the renal papillae and glomeruli determined. An additional five diabetic rats were treated with protamine zinc insulin for 1 week before sacrifice to determine the effect of insulin treatment on the production of PGE, PGF, and TxB by the glomeruli and renal papillae. Papillary production of PGE, PGF, and TxB was decreased in diabetic rats, as was glomerular production of PGE and PGF. Insulin treatment increased PGF production by the renal papillae and increased PGE, PGF, and TxB production by glomeruli. These results indicate that changes in prostaglandin excretion accompany the development of marked proteinuria and a decrease in creatinine clearance.(ABSTRACT TRUNCATED AT 250 WORDS)

Quantitative bacteriology and pathology of the lung in experimental Pseudomonas pneumonia treated with positive end-expiratory pressure (PEEP).

Mechanical ventilation with positive end-expiratory pre-sure (PEEP) is widely used to treat ventilatory failure complicating pulmonary infection. The present experiment was carried out to test the hypothesis that PEEP is beneficial in an experimental model of canine pneumonia studied for 24 hours. Sixteen mongrel dogs were assigned to ventilation with either zero end-expiratory pressure (ZEEP) or 10 cm H2O PEEP. Pneumonia was induced in half of each group by intratracheal inoculation with Pseudomonas. Tissues for quantitative bacteriology and pathology were obtained at the time of death at 24 hours. Three of four infected-ZEEP dogs died before 24 hours. The geometrical mean of quantitative bacterial counts from infected-ZEEP lobes was 2.0 X 10(6) (+/-3.9) (organisms/gm of tissue), while the mean of the infected-PEEP lobes was 1.7 X 10(4) (+/-3.6) (p less than 0.05). Semiquantitative pathology scores indicated greater injury to the ZEEP-infected than to the PEEP-infected lungs. Quantitative bacteriology and microscopic evidence of parenchymal injury were positively correlated. Thus PEEP-treated animals had lower quantitative bacterial counts, less microscopic pulmonary damage, and improved survival. The advantage conferred by PEEP may be due to facilitation of local mechanisms of pulmonary defense against infection, to increased systemic resistance to sepsis, or to both.