Longitudinal follow-up of CA125 in peritoneal effluent

Mesothelial changes occur during peritoneal dialysis. CA125 provides a way to study the mesothelial cells in the in vivo situation. In the present study longitudinal changes of CA125 were analyzed. In addition, the appearance of CA125 in peritoneal effluent and day-to-day variability were studied. CA125 was measured in the effluent of five stable CAPD patients during four hour dwells with 1.36% glucose, with 3.86% glucose and with 7.5% icodextrin. In addition, CA125 was determined on six consecutive days in four hour effluents of three patients and appearance rates (AR) were calculated. Longitudinal follow-up was performed in 31 patients in whom three to seven yearly observations had been made. Linear appearance of CA125 was present in all dwells. No difference was found between the appearance rates of CA125 with 3.86% glucose, compared to either 1.36% glucose or icodextrin. Mean day-to-day coefficient of variation was 6.4% for CA125 AR, but a wide variation existed in stable CA125 values among patients (mean 22.1, range 2 to 48 U/ml). A negative trend with duration of CAPD was present in the longitudinal study. A mean decrease of 2.2% per year could be calculated, but substantial interindividual differences existed. Sudden decreases of CA125 AR were found in five patients. Possible causes were found in all of them and included a severe or recurrent peritonitis, and temporary cessation of peritoneal dialysis. In one patient a sudden decrease preceded the manifestation of peritoneal sclerosis. It can be concluded that CA125 can be used for the in vivo follow-up of the mesothelium in peritoneal dialysis patients. The appearance of CA125 in effluent is linear in time and not influenced by the initial lysis of mesothelial cells. A gradual loss of mesothelial cells is likely to occur, although interindividual variability is substantial. An acceleration of the process may be caused by severe peritonitis and perhaps by temporary cessation of peritoneal dialysis. A sudden decrease in CA125 may be an alarming sign for the development or manifestation of peritoneal sclerosi

Markers of peritoneal mesothelial cells during treatment with peritoneal dialysis

Loss of peritoneal mesothelial cells and decrease of mesothelial cell mass have been described in peritoneal dialysis (PD) patients. Longitudinal follow-up in individual PD patients cannot be performed by serial peritoneal biopsies. Markers of mesothelial integrity, measured in the effluent, may therefore be a valuable approach to detect changes in the mesothelium in vivo. In the present study, markers that are known to be produced by mesothelial cells were followed in individual patients: cancer antigen 125 (CA125), phospholipids (PHL), and hyaluronan (HA). CA125 is considered to be a reflection of mesothelial cell mass or stable mesothelial cell turnover. Appearance rates (AR) were determined in the effluents of 30 PD patients on a yearly basis. Median AR (range) were: CA125: 111 U/min (10-610), PHL: 15 mg/min (3-46), HA: 666 mg/min (135-6200). Cross sectionally, the AR for CA125 was negatively related to duration of PD (r = -0.47, p < 0.0001) and weakly related to peritonitis incidence (r = -0.20, p < 0.05). Patients treated with PD for more than 4 years had lower CA125 appearance than patients treated less than 4 years (p < 0.0004). HA was also related to the incidence of peritonitis, but positively (r = 0.32, p < 0.004). PHL were not related to either parameter. A significant negative trend with time of PD treatment was observed for CA125 only [mean regression coefficient (t) -3.75, SD 1.2]. No trend in time of PD treatment could be detected for HA and PHL. These data indicate a gradual loss of mesothelial cell mass during PD by the decrease of CA125 with time. The lack of a decrease in HA and its positive relation to incidence of peritonitis suggest an additional release of HA by cells other than the mesothelial cells, such as fibroblasts and leukocytes. Alternatively, an activation of (mesothelial) cells with duration of PD and possibly with increased peritonitis incidence cannot be excluded. The relation between PHL and duration of PD suggested by others was not confirmed in this study. PHL are probably released by a number of different cells, and therefore changes in PHL cannot be used as a reflection of changes in mesothelial cell mass. It is concluded that CA125 is the most specific marker for the follow-up of mesothelial cell mass in viv

Icodextrin with nitroprusside increases ultrafiltration and peritoneal transport during long CAPD dwells

Addition of the nitric oxide (NO) donor nitroprusside to 1.36% glucose dialysate enlarges the effective peritoneal surface area during four-hour dwells. The theoretical positive effect on ultrafiltration is, however, counteracted by an increase in glucose absorption. The absorption of the glucose polymer icodextrin is much lower in comparison with glucose-based dialysis solutions, due to its high molecular weight. In the present study 7.5% icodextrin dialysis solution with and without the addition of 4.5 mg/liter nitroprusside was studied during eight-hour CAPD dwells. Two Standard Peritoneal permeability Analyses, adapted for eight-hour dwells, were performed in 10 stable CAPD patients. Nitrate and cGMP were measured as parameters of NO synthesis. The transcapillary ultrafiltration increased in a linear way with icodextrin (ICO) and was even higher after the addition of nitroprusside (NP): 666 (ICO) versus 834 (NP) ml/8 hr, P = 0.03. The effective lymphatic absorption rate was not different. The resulting net ultrafiltration increased with nitroprusside: 344 (ICO) versus 540 (NP) ml/8 hr, P < 0.01. The mass transfer area coefficient of urea increased 15% and that of creatinine 26% with nitroprusside, consistent with the expected enlargement of the vascular peritoneal surface area. The increase in protein clearances was more pronounced the larger the protein: beta 2-microglobulin 19%, albumin 47%, IgG 63% and alpha 2-macroglobulin 95%. Dialysate/plasma (D/P) ratios of nitrate were not higher than the expected values on the basis of its molecular weight (P < 0.001). They increased 19% with nitroprusside. Also, the D/P ratio cyclic guanosine monophosphate (cGMP) after four hours increased with nitroprusside (0.39, range 0.13 to 0.55 ICO, and 0.82, range 0.36 to 1.39 NP, P = 0.01). With nitroprusside the D/P ratio cGMP was higher than expected after four and eight hours (P < 0.001). This points to local generation of NO after addition of nitroprusside. The nitroprusside induced increase in the mass transfer area coefficients (MTAC) of creatinine and in the ultrafiltration caused an increase in the creatinine clearance from 4.2 ml/min to 5.0 ml/min during the eight-hour dwell. This means that nitroprusside adds 3 liters/week to the peritoneal clearance of creatinine. The adequacy of peritoneal dialysis can therefore be improved by the addition of nitroprusside to 7.5% icodextrin, used for the long exchang

Immune response to lentiviral bilirubin UDP-glucuronosyltransferase gene transfer in fetal and neonatal rats

Gene therapy for inherited disorders might cause an immune response to the therapeutic protein. A solution would be to introduce the gene in the fetal or neonatal period, which should lead to tolerization. Lentiviral vectors mediate long-term gene expression, and are well suited for gene therapy early in development. A model for fetal or neonatal gene therapy is the inherited disorder of bilirubin metabolism, Crigler-Najjar disease (CN). The absence of bilirubin UDP-glucoronyltransferase (UGT1A1) activity in CN patients causes high serum levels of unconjugated bilirubin and brain damage in infancy. CN is attractive for the development of gene therapy because the mutant Gunn rat closely mimics the human disease. Injection of UGT1A1 lentiviral vectors corrected the hyperbilirubinemia for more than a year in rats injected as fetuses and for up to 18 weeks in rats injected the day of birth. UGT1A1 gene transfer was confirmed by the presence of bilirubin glucuronides in bile. All animals injected with UGT1A1 lentiviral vectors developed antibodies to UGT1A1. Animals injected with green fluorescent protein (GFP) lentiviral vectors did not develop antibodies to GFP. Our results indicate that fetal and neonatal gene therapy with immunogenic proteins such as UGT1A1 does not necessarily lead to tolerizatio

Cellular Localization and Biochemical Analysis of Mammalian CDC50A, a Glycosylated beta-subunit for P4 ATPases

CDC50 proteins are beta-subunits for P4 ATPases, which upon heterodimerization form a functional phospholipid translocation complex. Emerging evidence in mouse models and men links mutations in P4 ATPase genes with human disease. This study analyzed the tissue distribution and cellular localization of CDC50A, the most abundant and ubiquitously expressed CDC50 homologue in the mouse. The authors have raised antibodies that detect mouse and human CDC50A and studied CDC50A localization and glycosylation status in mouse liver cells. CDC50A is a terminal-glycosylated glycoprotein and is expressed in hepatocytes and liver sinusoidal endothelial cells, where it resides in detergent-resistant membranes. In pancreas and stomach, CDC50A localized to secretory vesicles, whereas in the kidney, CDC50A localized to the apical region of proximal convoluted tubules of the cortex. In WIF-B9 cells, CDC50A partially costains with the trans-Golgi network. Data suggest that CDC50A is present as a fully glycosylated protein in vivo, which presumes interaction with distinct P4 ATPases. (J Histochem Cytochem 60: 205-218, 2012

Kupffer cells and not liver sinusoidal endothelial cells prevent lentiviral transduction of hepatocytes

Lentiviral vectors can stably transduce dividing and nondividing cells in vivo and are best suited to long-term correction of inherited liver diseases. Intraportal administration of lentiviral vectors expressing green fluorescent protein (Lenti-GFP) in mice resulted in a higher transduction of nonparenchymal cells than hepatocytes (7.32 +/- 3.66% vs 0.22 +/- 0.08%, respectively). Therefore, various treatments were explored to increase lentiviral transduction of hepatocytes. Lenti-GFP was injected into the common bile duct, which led to transduction of biliary epithelium and hepatocytes at low efficiency. Transient removal of the sinusoidal endothelial cell layer by cyclophosphamide to increase accessibility to hepatocytes did not improve hepatocyte transduction (0.42 +/- 0.36%). Inhibition of Kupffer cell function by gadolinium chloride led to a significant decrease in GFP-positive nonparenchymal cells (2.15 +/- 3.14%) and a sevenfold increase in GFP-positive hepatocytes compared to nonpretreated mice (1.48 +/- 2.01%). These findings suggest that sinusoidal endothelial cells do not significantly limit lentiviral transduction of hepatocytes, while Kupffer cells sequester lentiviral particles thereby preventing hepatocyte transduction. Therefore, the use of agents that inhibit Kupffer cell function may be important for lentiviral vector treatment of liver diseas

Phospholipid flippases attenuate LPS-induced TLR4 signaling by mediating endocytic retrieval of Toll-like receptor 4

P4-ATPases are lipid flippases that catalyze the transport of phospholipids to create membrane phospholipid asymmetry and to initiate the biogenesis of transport vesicles. Here we show, for the first time, that lipid flippases are essential to dampen the inflammatory response and to mediate the endotoxin-induced endocytic retrieval of Toll-like receptor 4 (TLR4) in human macrophages. Depletion of CDC50A, the β-subunit that is crucial for the activity of multiple P4-ATPases, resulted in endotoxin-induced hypersecretion of proinflammatory cytokines, enhanced MAP kinase signaling and constitutive NF-κB activation. In addition, CDC50A-depleted THP-1 macrophages displayed reduced tolerance to endotoxin. Moreover, endotoxin-induced internalization of TLR4 was strongly reduced and coincided with impaired endosomal MyD88-independent signaling. The phenotype of CDC50A-depleted cells was also induced by separate knockdown of two P4-ATPases, namely ATP8B1 and ATP11A. We conclude that lipid flippases are novel elements of the innate immune response that are essential to attenuate the inflammatory response, possibly by mediating endotoxin-induced internalization of TLR