Characterizing treatment resistance in muscle invasive bladder cancer using the chicken egg chorioallantoic membrane patient-derived xenograft model.

Background: Non-metastatic muscle invasive urothelial bladder cancer (MIBC) has a poor prognosis and standard of care (SOC) includes neoadjuvant cisplatin-based chemotherapy (NAC) combined with cystectomy. Patients receiving NAC have at best <10% improvement in five-year overall survival compared to cystectomy alone. This major clinical problem underscores gaps in our understanding of resistance mechanisms and a need for reliable pre-clinical models. The chicken embryo chorioallantoic membrane (CAM) represents a rapid, scalable, and cost-effective alternative to immunocompromised mice for establishing patient-derived xenografts (PDX) in vivo. CAM-PDX leverages an easily accessible engraftment scaffold and vascular-rich, immunosuppressed environment for the engraftment of PDX tumors and subsequent functional studies. Methods: We optimized engraftment conditions for primary MIBC tumors using the CAM-PDX model and tested concordance between cisplatin-based chemotherapy response of patients to matching PDX tumors using tumor growth coupled with immunohistochemistry markers of proliferation and apoptosis. We also tested select kinase inhibitor response on chemotherapy-resistant bladder cancers on the CAM-PDX using tumor growth measurements and immuno-detection of proliferation marker, Ki-67. Results: Our results show primary, NAC-resistant, MIBC tumors grown on the CAM share histological characteristics along with cisplatin-based chemotherapy resistance observed in the clinic for matched parent human tumor specimens. Patient tumor specimens acquired after chemotherapy treatment (post-NAC) and exhibiting NAC resistance were engrafted successfully on the CAM and displayed decreased tumor growth size and proliferation in response to treatment with a dual EGFR and HER2 inhibitor, but had no significant response to either CDK4/6 or FGFR inhibition. Conclusions: Our data suggests concordance between cisplatin-based chemotherapy resistance phenotypes in primary patient tumors and CAM-PDX models. Further, proteogenomic informed kinase inhibitor use on MIBC CAM-PDX models suggests a benefit from integration of rapid in vivo testing of novel therapeutics to inform more complex, pre-clinical mouse PDX experiments for more effective clinical trial design aimed at achieving optimal precision medicine for patients with limited treatment options.

Effect of cyclosporin A on DNA repair and cancer incidence in kidney transplant recipients.

Cancer incidence is enhanced in transplant recipients. Decreased DNA repair ability is associated with increased cancer incidence. Transplanted patients with cancer were found to have reduced DNA repair. We hypothesized that immunosuppressive therapy may impair DNA repair and thus contribute to the increased cancer incidence in transplanted patients. The objectives of this study were (1) to investigate the effect of two immunosuppressive treatment protocols on DNA repair in kidney transplant recipients; (2) to evaluate the cancer incidence in these patients; and (3) to study the in vitro effect of cyclosporin A (CsA), azathioprine, and prednisolone-separately and in various combinations-on DNA repair. Three groups were studied: (1) a control group; (2) patients treated with azathioprine and prednisone (double-therapy group); and (3) patients treated with CsA, azathioprine, and prednisone (triple-therapy group). The two patient groups did not differ in age, gender, time on dialysis before transplantation, or kidney function or in the number of acute rejections. However, the interval from transplantation to the DNA repair study was shorter in the triple-therapy group (P <.01). DNA repair was induced in peripheral blood mononuclear cells (PBMCs) by ultraviolet irradiation and expressed as tritiated thymidine uptake by these cells. DNA repair in the triple-therapy group was 679 +/- 64 cpm/10(6) cells, significantly less than that in the control group (1049 +/- 69 cpm/10(6) cells, P <.02). In the double-therapy group, DNA repair was similar to that in the control group. The follow-up period was shorter in the triple-therapy group (116 +/- 19 months vs 174 +/- 29 months, P <.01). Five tumors developed in the triple-therapy group, but only one developed in the double-therapy group (P =.05). The in vitro study showed a dose-dependent reduction in PBMC DNA repair by CsA. Azathioprine and prednisolone reduced DNA repair slightly, but CsA reduced DNA repair significantly more than either one or a combination of them. In summary, triple therapy was associated with impaired PBMC DNA repair and increased cancer incidence. CsA was responsible in large part for the reduction in DNA repair ability found in the in vitro and in vivo studies. This may have partly contributed to the enhanced cancer incidence in the kidney transplant recipients.

Haemolysis in haemodialysis patients: evidence for impaired defence mechanisms against oxidative stress.

BACKGROUND: Uraemic patients have a decreased ability to withstand oxidative stress. It is postulated that their antioxidant capacity is reduced, yet the mechanism remains unclear. Recently 33 haemodialysis (HD) patients were exposed to chloramine contamination in the water supply. This led to haemolysis in 24 patients, while nine were unaffected. In the former group haemoglobin decreased from 11.7+/-1.1 to 8.5+/- 1.4 g/dl (P<0.0001) and returned to 11.4+/-0.9 g/dl (P<0.0001) following recovery. During haemolysis, haptoglobin was 38.4+/-10.6 vs 138.1+/-8.3 ng/dl (P<0.0001) following recovery. METHODS: To explore the factors affecting the severity of haemolysis we studied extracellular and intracellular anti-oxidant defence mechanisms 3 months after recovery. In 29 patients and 20 controls we determined plasma glutathione (GSH), and the erythrocyte enzymes glutathione peroxidase (GSH-Px), glutathione reductase (GSH-Rx), and superoxide dismutase (SOD). Serum malondialdehyde (MDA) was measured as a marker of oxidative stress. RESULTS: Plasma GSH was lower in patients as compared to controls (5.49+/-0.26 vs 7.4+/-0.5 micromol/l, P<0.005). There was an inverse correlation between GSH and the degree of haemolysis (r=-0.42, P<0.02). Patients had higher GSH-Rx (4.64+/-0.15 vs 3.97+/-0.12 U/gHb, P<0.02), lower GSH-Px (29. 7+/-1.85 vs 35.5+/-1.62 U/gHb, P<0.001), and similar SOD (0.63+/-0. 02 vs 0.51+/-0.02 U/mgHb) as compared to controls. There was no correlation between the enzyme levels and the degree of haemolysis. MDA was higher in patients (2.37+/-0.07 vs 0.97+/-0.1 nmol/ml, P<0. 0001). There was a correlation between MDA and the years patients were on HD (r=0.43, P<0.02). CONCLUSIONS: These data indicate that HD patients have an impaired anti-oxidant response, which may be attributed in part, to plasma GSH deficiency. Patients with the lowest plasma GSH levels are more susceptible to oxidative stress and consequent haemolysis.

Lymphocytic intracellular calcium in a patient with complicated verapamil overdose.

Overdose with calcium channel blockers (CCBs) may lead to serious complications. CCBs act by blocking calcium entry into the cell, thus lowering intracellular calcium ([Ca2+]i). [Ca2+]i during CCB overdose has not yet been reported. We measured [Ca2+]i in lymphocytes of a patient with acute verapamil overdose with a complex clinical picture. A 59-year-old woman was admitted after a suicidal ingestion of 7200 mg of a sustained-release verapamil preparation. She presented with hypotension, complete atrioventricular block, stupor, hypokalemia, and hyperglycemia. Acute oliguric renal failure, acute pancreatitis, and the adult respiratory distress syndrome further complicated her medical course. Treatment was supportive and she recovered completely. Intracellular calcium ([Ca2+]i) was measured in the patient's lymphocytes using a spectrofluorometer with the calcium-sensitive dye Fura-2-acetoxymethyl ester. Thirty nine hours after the ingestion, [Ca2+]i was low at 52 nM (compared with 80 nM in a healthy control subject). Lymphocytic [Ca2+]i did not respond to stimulation with phytohemagglutinin (PHA). Fourteen days after the verapamil overdose, after the patient had recovered completely, lymphocytic [Ca2+]i was still low at 55 nM. At this time, there was an incomplete response to PHA in the lymphocytes. Three months after the ingestion, [Ca2+]i was normal, with a normal response to PHA. Verapamil overdose may run a complex clinical course, but full recovery is to be hoped for with full supportive care. Cellular intoxication, as reflected by low lymphocytic [Ca2+]i, is prolonged and lags behind the clinical recovery by weeks.

Erythropoietin, folic acid deficiency and hyperhomocysteinemia: is there a possible relationship in chronically hemodialyzed patients?

AIMS: To examine the possible relationships between recombinant human erythropoietin (rhEPO) therapy, serum folic acid and homocysteine levels in a cohort of stable, chronically hemodialyzed patients. MATERIAL AND METHODS: The study was cross-sectional in its first phase and consisted of 3 groups of subjects (group 1:6 healthy controls; group 2:7 dialyzed patients not receiving rhEPO; group 3: 14 patients on rhEPO therapy). Hematological and biochemical parameters were taken after an overnight fast in all subjects. The second phase of the study was prospective, and included 8 dialyzed patients, and investigated the effects of a 6-month period of folic acid supplementation (10 mg, 3 times a week) on the same parameters examined in the first phase of the study. RESULTS: In the first part of the study hemoglobin levels were near-normal, or normal, in all patients. No differences in hemoglobin or hematocrit values were observed in the 3 groups. 80% of all hemodialyzed patients had low serum folic acid levels, irrespective of whether they were receiving rhEPO. Serum erythropoietin level was elevated in group 3 (23.3+/-10.4 mIU/ml). In group 2, serum erythropoietin level was not different from that of the healthy controls (13.5+/-11.2 vs. 8.0+/-5.4 mIU/ml, p = n.s.). Total serum homocysteine levels were elevated in all dialyzed patients (group 2: 24.7+/-9.2 micromol/l; group 3: 31.6+/-14.4 micromol/l), with a significant difference seen when comparing controls and those dialyzed patients on rhEPO therapy (8.7+/-2.2 vs. 31.6+/-14.4 micromol/l; p<0.05). Significant correlations (ANOVA) were observed between serum erythropoietin and folic acid levels (r = -0.382; p = 0.049), and between folic acid and homocysteine levels (r = -0.560; p = 0.002). In the second part of the study folic acid supplementation led to a highly significant reduction in homocysteine levels (20.9+/-4.9 vs. 11.9+/-2.5 micromol/l; p<0.0005). Two of 3 patients receiving rhEPO therapy, had rhEPO discontinued after commencing folic acid, as hemoglobin levels remained adequate, even without rhEPO. CONCLUSIONS: In hemodialyzed patients, the presence of a near-normal hemoglobin level, irrespective of rhEPO therapy, implies efficient erythropoiesis. Without adequate folic acid reserves, folic acid deficiency may develop in these patients and this will aggravate already high homocysteine levels. Therefore, folic acid supplementation is warranted in hemodialyzed patients, especially in those patients with hemoglobin levels approaching normal. This treatment is safe and effective in reducing homocysteine levels, especially when given in high doses for prolonged periods of time.

H2O2 induces DNA repair in mononuclear cells: evidence for association with cytosolic Ca2+ fluxes.

Cellular DNA repair systems are induced whenever DNA is damaged. Reactive oxygen species (ROS) are generated, in vivo, in the tissues as a result of regular cellular metabolism or after exposure to oxidizing agents, such as ultraviolet (UV) irradiation. It has been suggested that ROS mediate DNA damage. The objectives of the study were as follows: (1) to investigate whether hydrogen peroxide (H2O2), the commonly occurring cellular ROS, induces DNA repair as a response to the damage it probably causes; (2) to evaluate whether H2O2-induced DNA repair, if present, is signaled through a Ca2(+)-dependent pathway via the tyrosine kinase signal transduction. H2O2 was found to induce DNA repair in human peripheral blood mononuclear cells (PBMCs) in a dose-dependent manner. The recovery of RNA synthesis, which occurred after DNA repair, confirmed that transcribable DNA was repaired. The inhibition of tyrosine kinase activity by genistein reduced the DNA repair significantly. Furthermore, H2O2 caused a dose-dependent significant rise in cytosolic calcium ((Ca2+)i). H2O2 also induced a small rise in (Ca2+)i of cytosolic Ca2(+)-depleted cells, probably reflecting the release of Ca2+ from internal stores. Genistein inhibited both Ca2+ influx and Ca2+ release from internal stores. In summary, H2O2 induced a DNA repair synthesis that was in part Ca2+ dependent and signaled via tyrosine kinase. The changes in DNA repair paralleled changes in (Ca2+)i. The H2O2-induced (Ca2+)i rise was mostly the result of influx, but to some degree it was also due to the translocation of Ca2+ from internal stores.

Impaired lymphocyte calcium metabolism in end-stage renal disease: enhanced influx, decreased efflux, and reduced response to mitogen.

Lymphocytes from patients with end-stage renal disease (ESRD) exhibit elevated cytosolic calcium concentration ((Ca2+)i), but the mechanisms responsible for this elevated (Ca2+)i have not been entirely elucidated. In addition, lymphocyte proliferative responses to mitogenic stimuli are suppressed in patients with ESRD. The objectives of the study were as follows: (1) to measure calcium influx and efflux in lymphocytes from patients with ESRD; (2) to measure the effect of the calcium regulator parathyroid hormone (PTH) on lymphocyte (Ca2+)i; (3) to measure cytosolic calcium signal in patients' lymphocytes after mitogenic stimulation. The three study groups were as follows: healthy subjects (control), patients with chronic renal failure (CRF) before the beginning of regular dialysis treatment, and patients undergoing regular hemodialysis (HD) treatment. Peripheral blood lymphocytes were tested in vitro for (Ca2+)i, Ca2+ influx, and membrane calcium-adenosine triphosphatase (CaATPase) activity. Cytosolic Ca2+ signals were traced after stimulations by PTH and by phytohemagglutinin (PHA). Baseline (Ca2+)i was significantly elevated in both ESRD groups. Ca2+ influx was enhanced and CaATPase activity was reduced in both ESRD groups. PTH caused a (Ca2+)i increase in normal cells in a dose-dependent manner. PHA caused a (Ca2+)i elevation, with a Ca2+ signal in both groups of patients with ESRD that was significantly smaller than that in the control group. These findings suggest that the high (Ca2+)i found in lymphocytes from patients with ESRD is the result of enhanced Ca2+ influx concomitant with reduced Ca2+ extrusion, as reflected by reduced CaATPase activity. The patients' elevated serum PTH levels may have contributed to the high (Ca2+]i. The impaired cytosolic (Ca2+)i response to PHA may explain in part the suppressed lymphocyte proliferative response to PHA in patients with ESRD.

The role of calcium in human lymphocyte DNA repair ability.

DNA repair ability is reduced in a variety of pathologic conditions. In addition, in some of these diseases a disturbance in cellular Ca homeostasis occurs or cytosolic (Ca2+) responses to various stimuli are impaired. The leading environmental cause for genomal DNA damage is ultraviolet (UV) irradiation. The aims of the present study were (1) to evaluate a possible dependence of UV-induced DNA repair ability on cytosolic Ca2+ in human lymphocytes and (2) to assess the direct effect of UV irradiation on Ca2+ homeostasis in these cells. UV-induced DNA repair ability in lymphocytes was maximal at 1 mmol/L CaCl2 in the medium. Suppression of DNA repair ability occurred after elevation or reduction of cellular (Ca2+) when various methods were used, including changes in Ca2+ concentration in the medium, cellular Ca2+ depletion by ethyleneglycol-bis-(beta aminoethylether)-N,N,N',N'-tetraacetic acid, excessive Ca2+ concentration induced by ionophore, and shortening of Ca2+ presence time during repair synthesis. UV irradiation caused an immediate and significant rise in cytosolic (Ca2+) that was the result of both enhanced Ca2+ uptake and inhibition of plasma membrane Ca-adenosine triphosphatase activity. The tyrosine kinase inhibitor genistein inhibited both UV-induced DNA repair and UV-induced cytosolic (Ca2+) elevation. These results emphasize the importance of a precise cellular Ca2+ level regulation for the optimal DNA repair process. UV irradiation, by inducing cellular Ca2+ rise, may activate DNA repair as soon as DNA is damaged.

Bullous dermatosis of end-stage renal disease: a possible association between abnormal porphyrin metabolism and aluminium.

BACKGROUND: Bullous dermatosis (BD) is becoming increasingly recognized in patients with end-stage renal disease (ESRD). It is clinically reminiscent of porphyria cutanea tarda, but its detailed pathogenesis remains unclear. Studies have shown increased porphyrin levels in dialysis patients, and this may partly explain the skin lesions and photosensitivity evident in these patients. In experimental studies, aluminum can induce various abnormalities in porphyrin and haem metabolism. This study investigated a possible involvement of porphyrin metabolism and aluminum in the development of bullous dermatosis in chronically dialysed patients. METHODS: Three groups were studied (12 healthy controls; 12 patients on chronic dialysis without BD and six patients on chronic dialysis with BD). Clinical characteristics of these patients were evaluated and the levels of plasma porphyrins, erythrocyte porphyrins and enzymes involved in the porphyrin chain were determined. RESULTS: The patients with BD were predominantly male, 50% had ADPKD, all had been on dialysis for a long period of time (7.8 +/- 2.1 years) and all were anuric. CAPD and haemodialysis were used equally in the affected patients. Aminolaevulinic dehydratase activity was significantly reduced in all ESRD patients (892 +/- 47 versus 302 +/- 36 versus 408 +/- 37 nmol/ml RBC/h). Plasma uroporphyrins as well as RBC protoporphyrin were significantly elevated in ESRD patients (1.7 +/- 0.6 versus 21.6 +/- 4.7 versus 43.4 +/- 12.0 nmol/L) and (1.43 +/- 0.14 versus 2.4 +/- 0.42 versus 4.19 +/- 2.44 mumol/l) respectively. Serum A1 levels were markedly elevated in patients with BD (28.3 +/- 10.0 micrograms/l). Both uroporphyrin and protoporphyrin were significantly more elevated in ESRD patients with BD compared to ESRD patients without BD. CONCLUSIONS: Elevated plasma porphyrin levels in ESRD patients are caused by lack of urinary excretion and the inability of haemodialysis and CAPD therapy to remove them. These elevated porphyrin levels may lead to the development of porphyria cutanea tarda symptoms. Elevations in plasma uroporphyrin, red blood cell protoporphyrin, and elevated A1 levels suggest a possible relationship between an A1 'load' and abnormal porphyrin metabolism in the development of overt skin disease in the dialysed patient.

DNA repair and recovery of RNA synthesis in uremic patients.

A high frequency of cancer appears among uremic patients. As depressed DNA repair ability is thought to be one of the causes for malignancy in cancer prone diseases, the present study was undertaken to examine DNA repair in uremic patients. Unscheduled DNA repair synthesis in peripheral lymphocytes was measured after both ultraviolet (UV) and gamma irradiations. In hemodialysis (HD) patients the repairs were normal, but in chronic renal failure (CRF) patients not yet on dialysis treatment, both UV- and gamma-induced DNA repair abilities were depressed to about 60% of the control. Recovery of RNA synthesis after UV irradiation followed the same pattern: it was reduced in CRF but normal in HD cells. When CRF lymphocytes were incubated in normal plasma, the UV-stimulated DNA repair improved to a nearly normal level, whereas incubation of normal cells in CRF plasma depressed their repair capacity to 70% of the initial level. These results suggest that a plasmatic substance such as the carcinogenic heterocyclic amines may be involved in the impairment of DNA repair in chronic renal failure.

Red blood cell calcium level is elevated in women: enhanced calcium influx by estrogens.

Red blood cell (RBC) calcium level had been found to be higher in women than in men. This study was designed to evaluate whether this is a general phenomenon and to elucidate a possible mechanism for a gender-related difference in RBC calcium levels. Differences in RBC calcium levels between women and men were examined in normal subjects, in patients with chronic renal failure (CRF) who were known to have elevated RBC calcium levels, and in female and male rats. RBC calcium level was higher in healthy women (6.1 +/- 0.5 mumol/L in women vs 4.4 +/- 0.3 mumol/L in men; p < 0.01), in women with CRF (45.8 +/- 11.8 mumol/L vs 15.4 +/- 1.1 mumol/L in men with CRF; p < 0.025) and women undergoing hemodialysis treatment (43.4 +/- 4.7 mumol/L vs 8.8 +/- 0.9 mumol/L in men undergoing hemodialysis p < 0.001). RBC calcium levels in female rats were also significantly higher than those in male rats. Ovariectomy reduced RBC calcium levels in female rats to those of male rats, whereas castration of male rats had no effect on RBC calcium levels. These in vivo findings suggest that the elevated RBC calcium level is associated with activity of female sex hormones. To investigate a possible mechanism, the in vitro effect of beta-estradiol on calcium 45 influx into RBCs and its effect on basal and calmodulin-stimulated Ca adenosine triphosphatase (CaATPase) activity in RBC membranes was determined. CaATPase activity was not affected by beta-estradiol at various concentrations and different incubation periods.(ABSTRACT TRUNCATED AT 250 WORDS)

Impaired DNA repair in patients with end-stage renal disease and its improvement with hemodialysis.

DNA repair following ultraviolet (UV)-induced DNA damage is decreased in some cancer-prone diseases. Cancer frequency in end-stage renal disease patients is higher than in the normal population. Therefore, DNA repair from UV-induced damage in lymphocytes was determined in 11 hemodialysis (HD) patients, 11 patients on continuous ambulatory peritoneal dialysis (CAPD), 10 patients with chronic renal failure (CRF) who had not yet been dialyzed and in 12 controls. UV irradiation at 254 nm was followed by 3H-thymidine incorporation in the presence of hydroxyurea. DNA repair synthesis in CAPD and CRF was significantly reduced to only 69 and 54% of the control, respectively, whereas the repair of HD patients was close to normal (86%, p = n.s.). The impaired ability to correct DNA damage may contribute to the increased cancer incidence in uremic patients.

Red blood cell calcium level in chronic renal failure: effect of continuous ambulatory peritoneal dialysis.

Red blood cell (RBC) calcium, calcium 45 influx, and calcium extrusion as indicated by Ca-stimulated, Mg-dependent adenosine triphosphatase (CaATPase) was determined in patients with chronic renal failure (CRF), patients with CRF receiving continuous ambulatory peritoneal dialysis (CAPD) treatment, and controls. Cell calcium, which in the controls was 5.5 mumol/L of cells, was elevated in patients with CRF--30.6 +/- 6.8 mumol/L of cells (p less than 0.002)--and in patients receiving CAPD-23.6 +/- 6.7 mumol/L of cells (p less than 0.02). Basal CaATPase activity in controls was 850.7 +/- 66.7 nmol inorganic phosphate per milligram of protein per hour. It was suppressed in patients with CRF and patients receiving CAPD: 504.9 +/- 34.4 nmol inorganic phosphate per milligram of protein per hour and 618.2 +/- 47.3 nmol inorganic phosphate per milligram of protein per hour, respectively (p less than 0.01). Calmodulin-stimulated CaATPase revealed a pattern similar to that of CaATPase basal activity. RBC calcium showed an inverse correlation with CaATPase activity (r = -0.935, p less than 0.005) in patients with CRF. Calcium influx was increased in patients with CRF and in patients receiving CAPD: 12.00 +/- 1.34 mumol/L of cells per hour and 13.60 +/- 1.70, respectively, compared with 4.61 +/- 0.39 mumol/L of cells per hour in controls (p less than 0.001). Patients with CRF have elevated RBC calcium levels mainly related to decreased extrusion and to increased influx. CAPD fails to improve substantially these abnormalities. Plasma vanadium levels were markedly elevated in patients undergoing hemodialysis and marginally in patients receiving CAPD.(ABSTRACT TRUNCATED AT 250 WORDS)

Red blood cell calcium homeostasis in patients with end-stage renal disease.

Low cell calcium level is essential for preservation of red blood cell (RBC) membrane deformability and survival. RBCs from patients with end-stage renal disease (ESRD) demonstrate reduction in membrane deformability, possibly as a result of increased RBC cellular calcium level. To evaluate calcium homeostasis in RBCs from patients with ESRD, we measured cell calcium level, basal and "calmodulin"-stimulated calcium-stimulated Mg-dependent ATPase (CaATPase) activity, and calcium 45 efflux were measured before and after hemodialysis. The in vitro effect of uremic plasma and of urea on CaATPase activity of normal RBCs was tested, and 45Ca influx into RBCs of patients undergoing hemodialysis also was determined. A morphologic evaluation of red cells from patients with ESRD was performed with a scanning electron microscope. RBC calcium level in patients (mean +/- SEM 21.2 +/- 2.8 mumol/L of cells; n = 28) was higher than in controls (4.9 +/- 0.3 mumol/L of cells; n = 24; p less than 0.001). Hemodialysis had no effect on cell calcium level. Both basal and "calmodulin"-stimulated RBC CaATPase activities in patients with ESRD (n = 9) were reduced by approximately 50% (p less than 0.01), but after hemodialysis, enzyme activity returned to normal. 45Ca efflux from calcium-loaded cells, which was 2574.0 +/- 217.0 mumol/L of cells per 0.5 hours before hemodialysis, increased to 3140.7 +/- 206.8 mumol/L of cells per 0.5 hours after hemodialysis (p less than 0.005). In vitro incubation of normal RBCs with uremic plasma depressed CaATPase activity, but incubation with urea had no effect. RBCs of patients with ESRD revealed increased 45Ca influx, 7.63 +/- 1.15 mumol/L of cells per hour versus 4.61 +/- 0.39 mumol/L of cells per hour (p less than 0.025). RBCs of patients revealed a high incidence of spherocytosis and echynocytosis, which correlated with a high cell calcium level (r = 0.894, p less than 0.01). These results indicate that RBC calcium level is elevated in patients with ESRD and suggest that a dialyzable uremic factor inhibits RBC CaATPase activity and thereby calcium efflux, which may account for the elevated cell calcium level. The increased calcium influx further increases cellular calcium level. These abnormalities are associated with spherocytosis and echynocytosis and may contribute to the shortened survival of RBCs in uremia.

Sialyltransferase activity and sialic acid levels in multiple myeloma and monoclonal gammopathy.

A significant elevation of peripheral blood mononuclear cell sialyltransferase activity (STA) was observed in 14 patients with multiple myeloma (MM), and compared to 7 patients with monoclonal gammopathy of undetermined significance (MGUS) and to 10 controls. Serum sialyltransferase was significant higher in MM patients as compared to controls. It was also higher than in MGUS patients, but the difference here was not statistically significant. STA was also determined in mononuclear bone marrow cells of 5 patients with MM (with 50 to 100% plasma cells in the bone marrow aspirate) and found to be 19 times higher than that of bone marrow mononuclear cells from 6 patients with non-malignant disorders (with less than 1% plasma cells in the bone marrow aspirate). No significant differences were observed in peripheral blood mononuclear cell sialic acid levels between MM, MGUS and controls.

Sialic acid and neuraminidase activity in rat kidneys 6 months after uninephrectomy.

Sialic acid and neuraminidase activity were determined in the cortex of the remnant kidneys of six uninephrectomized rats. As controls served either the kidneys removed at operation or age-matched kidneys from eight sham operated rats. Six months after uninephrectomy the kidneys became hypertrophied and their mean weight was about 40% higher than age-matched kidneys. Blood urea and creatinine and protein levels in 24-hr urine collections were significantly higher in the experimental animals as compared to those of the same animals before nephrectomy and to sham operated rats, indicating a marked impairment of kidney function. The mean concentration of sialic acid in the cortex of hypertrophied kidneys was not statistically different from either that of the removed or that of the age-matched kidneys. Neuraminidase activity expressed as either per gram fresh tissue or per milligram protein, was not different in the removed and in the hypertrophied kidney. The activity, however, in the latter was significantly lower than in the age-matched kidneys. Whether this finding can be associated with the impairment of kidney function in rats 6 months after uninephrectomy remains to be studied.

Biochemical changes associated with the osmotic fragility of young and mature erythrocytes caused by parathyroid hormone in relation to the uremic syndrome.

The effect of parathyroid hormone at concentrations found in uremic patients on erythrocytes (RBC) from newborn and adult rabbits was studied in relation to the fragility pattern in hypotonic salt solutions and the activities of Ca- and Mg-dependent ATPases. Median osmotic fragility of RBC from newborn rabbits was significantly lower than in mature rabbits. Parathyroid hormone (PTH) stimulated to a greater extent the mean osmotic fragility in RBC from newborn rabbits, than in those from adults. Similarly, the hormone stimulated to a much greater extent the Ca-ATPase but not the Mg-ATPase in RBC from the newborn rabbits, in comparison to those from adult rabbits. PTH, which is greatly elevated in the blood of patients with chronic renal failure, may be one cause of the anemia seen in these patients, and its effect, which is mediated by Ca-ATPase activity, is stronger on young RBC. There were significant morphological changes in the young RBC caused by PTH, as seen with scanning electron microscopy.

Platelet count and thrombopoietic activity in patients with chronic renal failure.

The frequency of thrombocytopenia in patients with chronic renal failure (CRF) is controversial. This study was undertaken to investigate the platelet count in 55 patients with end-stage renal disease on maintenance hemodialysis and in 19 patients with CRF before hemodialysis had begun. In both groups platelet counts were similar and significantly reduced, 175,000 +/- 6,500 and 181,000 +/- 10,800 compared to 253,000 +/- 3,700/mm3 in the control (p less than 0.0001). 31% of hemodialysis patients had thrombocytopenia (platelet count less than 150,000/mm3). The megakaryocyte number in their bone marrow aspirate was not reduced. Primary renal disease, androgen treatment or parathyroidectomy did not affect the platelet count. Thrombopoietic activity using 75Se-selenomethionine incorporation into platelets measured in 7 thrombocytopenic patients was found to be reduced, 6.77 +/- 0.29 vs. 9.06 +/- 0.27 (X 10(-2)%: p less than 0.001). This study shows that the platelet count is reduced and mild thrombocytopenia is frequent in patients with CRF. A possible cause for the platelet count reduction is insufficient thrombopoietic activity.