Influence of uremia on cell viability and cytokine release of human peritoneal mesothelial cells.

INTRODUCTION: There is still no evidence whether human peritoneal mesothelial cells (HPMC) from patients with end-stage renal failure are altered in cell viability or show a different pattern of the release of proinflammatory cytokines. Also the serum of patients with uremia may contain substances stimulating the cytokine release of HPMC. STUDY DESIGN: The IL-1beta-induced IL-6/IL-8 release of HPMC from healthy donors and from patients with end-stage renal disease (ESRD) were measured before the start of chronic peritoneal dialysis (PD) and during PD therapy. Additionally the influence of uremic and non-uremic serum on IL-6 and IL-8 release of normal HPMC was studied. Cell viability was assessed by MTT assay and by the measurement of intracellular ATP (chemoluminescence assay). HPMC were obtained from the following patient groups: (1) non-uremic control patients (n = 7); (2) patients with ESRD undergoing PD catheter implantation for the first time (n = 7), and (3) patients on PD undergoing catheter exchange for noninfectious reasons (n = 6). Pooled human serum from PD patients and normal controls were used for stimulation experiments. HPMC from different donors were grown to confluence (second passage) and then stimulated with IL-1beta (1,000 pg/ml in M199) for 24 h. IL-6 and IL-8 concentrations were measured in the supernatant by ELISA. Additionally uremic and non-uremic sera were incubated with HPMC from normal donors for 24 h with a subsequent 24-hour IL-1beta stimulation. Mesothelial cell protein mass was determined by the Bradford reagent. RESULTS: Non-uremic patients and ESRD patients did not differ with regard to the global cell viability of HPMC according to MTT assay activity or the intracellular ATP concentration. However, HPMC from uremic patients produced more IL-8 on IL-1beta stimulation than the non-uremic controls (group 2, 53.5 +/- 15.7 pg/microg; group 3, 70.5 +/- 27.3 pg/microg vs. group 1, 24.0 +/- 11.8 pg/microg). HPMC from patients on chronic PD additionally released significantly more IL-6 (30.5 +/- 13.8 pg/microg) on IL-1beta stimulation than uremic patients before the onset of PD (6.2 +/- 2.6 pg/microg; p < 0.01). Incubation of normal HPMC with the serum from uremic donors produced an enhanced stimulated IL-8 release compared to the exposition with normal control serum (50.6 +/- 6.1 vs. 20.8 +/- 2.9 pg/microg; p < 0.01). CONCLUSION: HPMC from uremic patients more readily release IL-8 on stimulation with IL-1beta. On chronic PD treatment IL-6 release was further enhanced. Not further classified serum components in uremia also enhance IL-6 and IL-8 release of HPMC.

Effects of candesartan and perindopril on renal function, TGF-beta1 plasma levels and excretion of prostaglandins in stable renal allograft recipients.

AIMS: Although on account of their nephroprotective effects, ACE inhibitors and angiotensin receptor antagonists appear to be advantageous for patients after renal transplantation, their use in these patients has been limited up to now. This is in part due to the risk of inducing a decrease in the glomerular filtration pressure gradient with subsequent impairment of allograft function. The aim of the present study was to investigate the effects of ACE inhibitors and angiotensin receptor antagonists on renal function, excretion of prostaglandins as a parameter of glomerular hemodynamics and TGF-beta1 plasma levels during an 8-week withdrawal phase in pretreated patients. PATIENTS AND METHODS: Sixteen patients with stable long-term allograft function undergoing therapy with candesartan (group 1) and 16 patients with stable long-term allograft function undergoing therapy with perindopril (group 2) were included in the study. Any signs of chronic allograft dysfunction were defined as exclusion criteria. Renal function, albuminuria, TGF-beta1 plasma levels as well as the excretion of thromboxane B2 and 6-keto-prostaglandin-F-1alpha were monitored during an 8-week withdrawal phase of the angiotensin receptor antagonist or ACE inhibitor, respectively. Normotension was maintained throughout the study period through adjustment of other anti-hypertensive drugs. RESULTS: Creatinine clearance as well as TGF-beta1 plasma levels and the excretion of prostaglandins remained unchanged after discontinuation of candesartan or perindopril. However, after withdrawal of the substances a significant increase in albuminuria was noted in both patient groups throughout the observation period. After 8 weeks, median albuminuria had increased by 63% in group 1 and by 163% in group 2. CONCLUSIONS: We were able to demonstrate that the use of ACE inhibitors and angiotensin receptor antagonists in patients after renal transplantation is safe. Favorable effects of both substances on albuminuria were detectable in patients who showed no signs of chronic allograft dysfunction according to the usual criteria. Therefore, a nephroprotective effect of candesartan as well as of perindopril, is highly probable in patients after renal transplantation. Further investigations regarding routine use in these patients are therefore mandatory.

Peritoneal dialysis fluids with a physiologic pH based on either lactate or bicarbonate buffer-effects on human mesothelial cells.

Conventional lactate (Lac)-buffered peritoneal dialysis (PD) solutions have turned out to be detrimental to human peritoneal cells, especially because of a low pH. In the present study, we focus on potential differences between Lac and bicarbonate (Bic) as a buffer when adjusted to a physiological pH. All test fluids were buffered with either 40 mmol/L of Lac or 34 mmol/L of Bic, sterile filtered, and adjusted to a pH of 7.4. Osmotic agents used were 1.36% glucose (Glu), 3.86% Glu, 1% amino acids (AA), and 7.5% Glu polymer (Glupoly). Human peritoneal mesothelial cells (HPMCs) were isolated from the omentum majus, grown to confluence, and incubated after the second passage for 15 minutes (37 degrees C and 5% carbon dioxide) with the test fluids. Cytotoxicity was controlled by measuring apoptotic and necrotic cells with cytofluorometry. Aerobic cell metabolism (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide [MTT] assay) and intracellular adenosine triphosphate (ATP) concentrations were measured to assess cell viability. Release of interleukin-6 (IL-6) from HPMCs was determined as a parameter of cellular host defense. No significant difference in apoptosis or necrosis rates was found between the solutions adjusted to normal pH. However, in the MTT assay, Bic solutions were superior to corresponding Lac pendants at an identical pH of 7.4 (P < 0.01). Intracellular ATP concentrations reflected a very similar pattern (P < 0.05). Glupoly in combination with Lac showed an impaired pattern with both the MTT and ATP assays. Regarding IL-1beta-stimulated IL-6 release, there was a small, but not significantly better, response for Bic. Differences in manifest cell cytotoxicity reflected by apoptosis and necrosis rates could not be detected comparing PD solutions buffered with Lac or Bic at a physiological pH. However, distinct parameters of cell metabolism were superior with Bic compared with Lac. Especially Glupoly was inferior in combination with Lac as a buffer.

Peritoneal sclerosis in peritoneal dialysis patients related to dialysis settings and peritoneal transport properties.

BACKGROUND: Peritoneal sclerosis is a histopathologic finding that is probably causative of long-term system failure in peritoneal dialysis (PD). It may be due to uremic toxicity and the influence of peritoneal dialysate components. We intended to clarify to which degree peritoneal fibrosis and vascular changes were associated with the modalities of PD and the peritoneal transport characteristics. METHODS: Peritoneal biopsies of 41 patients suffering from end-stage renal disease were examined. Sixteen patients were at the initiation of dialysis treatment, and 25 patients were studied during chronic PD treatment [9 patients on continuous ambulatory PD (CAPD) and 16 patients on automatic PD (APD)]. Twenty nonuremic patients undergoing abdominal surgery served as the controls. Samples were taken from the parietal peritoneum under standardized conditions and were examined by light microscopy using different staining techniques, semiquantitative grading, and computer-based histomorphometry. RESULTS: A marked loss of mesothelial cells during PD treatment was only observed in cases with two or more preceding episodes of peritonitis, but an increase of the submesothelial fibrous tissue was a common finding and was related to the cumulative glucose load on PD. Patients on PD also had a significantly increased density of small vessels and capillaries in the submesothelial peritoneal layer (12.8 +/- 6.9 per field vs. 6.6 +/- 2.9 in normal controls, P < 0.01). The wall/lumen index of the vessels was increased indicating vascular sclerosis. The degree of vascularization correlated with the amount of fibrous tissue. Patients characterized as high transporters according to the peritoneal equilibration test (PET) had an increased submesothelial fibrous layer. Patients on APD tended to have an increased membrane diameter submesothelial stroma and vascularization (P = NS), although they were treated for a shorter period of time than the CAPD group. Some of the morphologic changes described could already be observed in uremic patients before the onset of dialysis. CONCLUSION: Further research focusing on the clinical and biochemical backgrounds leading to peritoneal membrane changes is of major importance for developing strategies to improve long-term survival on PD.

In vivo and in vitro effects of amino-acid-based and bicarbonate-buffered peritoneal dialysis solutions with regard to peritoneal transport and cytokines/prostanoids dialysate concentrations.

BACKGROUND: Increasing evidence suggests that conventional PD solutions are detrimental to host defence mechanisms of peritoneal cells. We tested a new amino-acid-based and bicarbonate-buffered PD solution under in vivo and in vitro conditions. METHODS: During a prospective, cross-over randomized, intraindividual study 10 CAPD patients were investigated with three different solutions: Amino/Bic, 1% amino acid, 34 mmol/l bicarbonate; Glu/Bic, 1.5% glucose, 34 mmol/l bicarbonate; and Glu/Lac, 1.5% glucose, 35 mmol/l lactate. A PET was performed and transport properties (clearance, D/P ratio, MTAC) were calculated. Prostanoid and cytokine concentrations were measured in serum and the 6 h effluent. Using an in vitro model, mononuclear leukocytes of healthy donors were also incubated with the test fluids. In vivo results. Peritoneal clearance and MTAC of small solutes (creatinine, urea) were not significantly altered by amino acids or bicarbonate. Peritoneal permeability and transperitoneal excretion of higher-weight protein molecules (beta 2-microglobulin, albumin, IgG) were increased with Amino/Bic compared to Glu/Lac (P < 0.05) (D/P ratio albumin: Amino/Bic, 0.027 +/- 0.003; Glu/Bic, 0.023 +/- 0.003; Glu/Lac, 0.022 +/- 0.002). Application of Amino/Bic was accompanied by an increased effluent concentration of Il-6, Il-8, TNF alpha, PGE2, and 6-keto-PGF1a (P < 0.05). Dialysate nitrite/nitrate and cGMP concentrations (as indicators of NO generation) did not differ between the solutions. In vitro results. Both bicarbonate fluids demonstrated a better preservation of the mitochondrial dehydrogenases activity (MTT assay) compared to Glu/ Lac (P < 0.01) (Amino/Bic: 80.6 +/- 3.2%; Glu/Bic: 86.0 +/- 1.8%; Glu/Lac, 64.9 +/- 2.3%, referred to RPMI as control). Constitutive and LPS stimulated release of Il-1 beta and Il-6 was less suppressed with both bicarbonate fluids (P < 0.05) (LPS-stim. Il-6 release: Amino/Bic, 33.0 +/- 6.6%; Glu/Bic, 65.5 +/- 10.3%; Glu/Lac, 1.5 +/- 0.7% referred to RPMI). CONCLUSION: Application of an amino-acid/bicarbonate solution resulted in a small but significant increase in peritoneal permeability. Also increased concentrations of various cytokines/prostanoids were measured in the effluent. According to in vitro testing with mononuclear phagocytes both bicarbonate-buffered fluids were to the same extent less inhibitory to certain cell functions than lactate-buffered solution.