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Janusz Witowski

Publications and source records attributed to Janusz Witowski.

16 recordsLinked to original sources
PubMed2005

Early loss of proliferative potential of human peritoneal mesothelial cells in culture: the role of p16INK4a-mediated premature senescence.

Much has been learned about the mechanisms underlying cellular senescence. The pathways leading to senescence appear to vary, depending on the cell type and cell culture conditions. In this respect, little is known about senescence of human peritoneal mesothelial cells (HPMC). Previous studies have significantly differed in the reported proliferative lifespan of HPMC. Therefore, in the present study, we have examined how HPMC enter state of senescence under conditions typically used for HPMC culture. HPMC were isolated from omentum and grown into senescence. The cultures were assessed for the growth rate, the presence of senescence markers, activation of cell-cycle inhibitors, and the oxidative stress. HPMC were found to reach, on average, six population doublings before senescence. The terminal growth arrest was associated with decreased expression of Ki67 antigen, increased percentage of cells in the G1 phase, reduced early population doubling level cDNA-1 mRNA expression, and the presence of senescence-associated beta-galactosidase. Compared with early-passage cells, the late-passage HPMC exhibited increased expression of p16INK4a but not of p21Cip1. In addition, these cells generated more reactive oxygen species and displayed increased presence of oxidatively modified DNA (8-hydroxy-2'-deoxyguanosine). These results demonstrate that early onset of senescence in omentum-derived HPMC may be associated with oxidative stress-induced upregulation of p16INK4a.

Blotting, Western↗
PubMed2005

Lessons from basic research for PD treatment.

Over the past 30 years, the focus of peritoneal dialysis research has changed from the technical issues related to the establishment of clinical peritoneal dialysis to complex problems of peritoneal membrane biology. Here, we present how these research topics developed, discuss their significance for clinical science, and outline future challenges for peritoneal dialysis research.

Dialysis Solutions↗
PubMed2004

L-2-oxothiazolidine-4-carboxylic acid reduces in vitro cytotoxicity of glucose degradation products.

BACKGROUND: Glucose degradation products (GDP) are an important factor that contribute to bioincompatibility of peritoneal dialysis fluids. These substances are generated in the dialysis fluid during heat sterilization. Several approaches have been proposed to reduce the content or toxicity, or both, of GDP present in the dialysis fluid. We examined whether L-2-oxothiazolidine-4-carboxylic acid (OTZ), a precursor for glutathione synthesis, reduces the cytotoxicity of GDP in human peritoneal mesothelial cells. METHODS: Experiments were performed on primary mesothelial cell cultures. Free radical generation in these cells after exposure to acetaldehyde (ACT), glyoxal (GLYO) or methylglyoxal (M-GLYO) was detected with a fluorescent probe. Cell viability measurements were based on release of LDH from cell cytosol, and synthesis of IL-6 and proliferation after exposure to GDP. Effects of individual GDPs and of dialysis fluid free of GDP (GDP-free PDF) or containing GDP (GDP-high PDF) on cell viability were also studied in the presence of OTZ (1 mmol/l). RESULTS: All of the GDPs as well as the autoclaved dialysis fluid caused increased free radical generation. ACT increased LDH release from the cells by 374% (P < 0.001), and this effect was abolished by OTZ. All of the GDPs inhibited cell growth (ACT, 47%, P < 0.01; GLYO, 52%, P < 0.01; M-GLYO, 26%, P < 0.05) and this effect was reversed in presence of OTZ. ACT inhibited Il-6 synthesis in mesothelial cells by 74% P < 0.01 and this effect was prevented by OTZ. GDP-high PDF but not GDP-free PDF reduced synthesis of IL-6 in mesothelial cells by 40% (P < 0.01) an effect that was reversed by OTZ. Mesothelial cell growth was more strongly inhibited by GDP-high PDF (76%, P < 0.01) than by GDP-free PDF (31%, P < 0.05). OTZ improved growth of mesothelial cells in the presence of GDP-high PDF (+150%, P < 0.01) and in presence of GDP-low PDF (+38%, P < 0.05). OTZ prevented the cytotoxic effect of GDP-high PDF on mesothelial cells. CONCLUSIONS: The GDP-induced stimulation of free radicals in mesothelial cells in the present study may provide a possible mechanism of GDP cytotoxicity. Because OTZ reduced the toxic effects of GDP on mesothelial cells, this compound may improve biocompatibility of peritoneal dialysis fluids.

Cell Division↗
PubMed2004

Peritoneal dialysis with solutions low in glucose degradation products is associated with improved biocompatibility profile towards peritoneal mesothelial cells.

BACKGROUND: In vitro experiments point to a better biocompatibility profile of new pH-neutral peritoneal dialysis fluids (PDFs) containing low levels of glucose degradation products (GDPs). The present study examines the impact on human peritoneal mesothelial cells (HPMCs) of equilibrated dialysates obtained during dialysis with either conventional or new PDFs. METHODS: Peritoneal dialysate was collected from 17 patients participating in a randomized, controlled, cross-over trial comparing a pH-neutral low-GDP solution (Balance) to a conventional solution (S-PDF). All patients were treated sequentially for 3 months with both PDFs. At the end of each treatment phase, peritoneal effluent was drained after a timed 10 h dwell. Samples of dialysate were then mixed with standard culture medium and added to in vitro cultures of HPMCs from healthy donors. Cells were assessed for proliferation, viability and cytokine release. RESULTS: Proliferation and viability of HPMCs were better preserved in the presence of effluent obtained during dialysis with Balance (P<0.046 and P<0.035, respectively). The proliferative response of HPMCs correlated with the concentration of fibronectin in dialysates (P = 0.0024). Effluent drained following a 3 month dialysis with Balance contained significantly increased levels of fibronectin (P = 0.004) and CA125 antigen (P = 0.0004) compared with S-PDF. There was no significant difference in constitutive and stimulated cytokine (IL-6, MCP-1, VEGF) synthesis by HPMCs treated with either Balance- or S-PDF-derived effluents. CONCLUSIONS: These results suggest that therapy with new pH-neutral low-GDP solutions contribute to an intraperitoneal milieu that improves mesothelial cell proliferation and viability. It may positively impact on the preservation of the peritoneal membrane integrity during long-term dialysis.

Adult↗
PubMed2003

Glucose-mediated induction of TGF-beta 1 and MCP-1 in mesothelial cells in vitro is osmolality and polyol pathway dependent.

BACKGROUND: Glucose is converted to sorbitol and then to fructose via the polyol pathway that has been implicated in the pathogenesis of organ damage. The contribution of the polyol pathway to mesothelial cell activation has, however, not been fully determined. METHODS: The effect of increasing glucose concentrations on transforming growth factor-beta 1 (TGF-beta 1) and monocyte chemoattractant protein-1 (MCP-1) secretion by human peritoneal mesothelial cells (HPMC) was examined. The importance of the polyol pathway was identified by its specific inhibition with an aldose reductase inhibitor. RESULTS: Incubation of HPMC with 5 to 100 mmol/L glucose resulted in an induction of aldose reductase mRNA and intracellular sorbitol accumulation accompanied by the induction of TGF-beta 1 and MCP-1 mRNA expression and protein secretion. Mannitol at the same concentrations also induced aldose reductase, TGF-beta 1 and MCP-1 mRNA and protein expression but at a lower level than glucose. Sorbinil dose-dependently reduced both intracellular sorbitol levels (79.8% reduction of 60 mmol/L D-glucose induced intracellular sorbitol with 100 micromol/L sorbinil (N = 3, P < 0.01) and glucose-induced TGF-beta 1 and MCP-1 secretion. Mannitol induced TGF-beta 1 and MCP-1 secretion was not reduced by sorbinil. The addition of 15 to 40 mmol/L sodium lactate, either alone or in the presence of D-glucose enhanced TGF-beta 1 and MCP-1 secretion, which was inhibited by sorbinil. In contrast, sodium pyruvate appeared to antagonize D-glucose-induced TGF-beta 1 and MCP-1 secretion. CONCLUSION: These data suggest that the polyol pathway and osmolality contribute to the regulation of HPMC function by glucose. Control of polyol pathway activation might reduce glucose-mediated damage to the peritoneal membrane and promote its long-term survival.

Aldehyde Reductase↗
PubMed2003

Glucose degradation products in peritoneal dialysis fluids: do they harm?

BACKGROUND: Severe limitations in biocompatibility of conventional peritoneal dialysis fluids (PDF) can be partially attributed to the presence of glucose degradation products (GDP), which are generated during autoclaving of PDF. Formation of GDP can be significantly reduced by the use of multi-chamber bag systems. Recent clinical studies have revealed increased dialysate levels of pro-collagen I C-terminal peptide (PICP) in patients dialyzed with these solutions. Here, we briefly review the current knowledge on various aspects of GDP toxicity toward peritoneal cells and analyze the impact of GDP on PICP release by human peritoneal mesothelial cells (HPMC) in vitro. METHODS: HPMC were exposed to a mixture of known GDP added to culture medium at clinically relevant doses. After 12 days, the amount of PICP released was measured using an immunoassay. Furthermore, the protein synthesis was assessed by 3H-proline incorporation in HPMC exposed to peritoneal effluent obtained from patients after three months of CAPD with either conventional PDF or low-GDP solution. RESULTS: Exposure to GDP resulted in a significant decrease in PICP release by HPMC. In addition, the synthesis of new proteins secreted by HPMC was preserved significantly better in HPMC treated with effluent obtained when patients were dialyzed with low-GDP solutions rather than conventional PDF. CONCLUSIONS: Exposure to GDP may impair protein synthesis and secretion by HPMC. Therefore, increased dialysate PICP levels in response to GDP-free PDF may be viewed as evidence of improved mesothelial cell function.

Dialysis Solutions↗
PubMed2003

Interplay between IFN-gamma and IL-6 signaling governs neutrophil trafficking and apoptosis during acute inflammation.

Regulated recruitment and clearance of neutrophils (PMN) is the hallmark of competent host defense and resolution of inflammation. We now report that IFN-gamma controls PMN infiltration and modulates IL-6 signaling through its soluble receptor (sIL-6R) to promote their apoptosis and clearance. Induction of peritoneal inflammation in IFN-gamma-deficient (IFN-gamma-/-) mice emphasized that the initial rate of PMN recruitment was impaired. This defect in PMN recruitment was also associated with the suppressed intraperitoneal expression of IL-1beta and IL-6. Reconstitution of IFN-gamma signaling restored the rate of PMN infiltration and IL-6 levels and was accompanied by normalization of PMN-activating CXC chemokine expression. To test whether local IL-6 signaling modulated PMN recruitment, inflammation was induced in IFN-gamma-/- and IL-6-/- mice and cytokine signaling adapted by intraperitoneal sIL-6R-IL-6 fusion protein (HYPER-IL-6) or IFN-gamma. Although HYPER-IL-6 attenuated PMN influx in IFN-gamma-/- mice, IFN-gamma had no effect on PMN infiltration in IL-6-/- mice. Examination of the leukocyte infiltrate from IFN-gamma-/-, IL-6-/-, and wild-type mice showed that apoptosis was aberrant in the absence of IFN-gamma and IL-6 as a result of impaired sIL-6R signaling. These data emphasize a pivotal role for IFN-gamma in regulating innate immunity through control of both the recruitment and clearance phases of PMN trafficking.

Animals↗
PubMed2002

[Dual role of nitric oxide in the pathogenesis of diabetic nephropathy].

Nitric oxide (NO) is a key mediator in renal physiology and pathology. In diabetic nephropathy, NO may exert destructive effects (hyperfiltration, peroxynitrate-mediated tissue injury) as well as exhibit certain protective properties (reduced TGF-beta expression and extracellular matrix expansion, inhibition of platelet aggregation). Here, we briefly review the biochemistry and pathophysiology of NO, and discuss the mechanisms underlying the contrasting effects of NO in diabetic nephropathy.

Diabetic Nephropathies↗
PubMed2002

[Aquaporin water channels in water balance regulation in the kidney].

The aquaporins (AQP) are a family of small transmembrane water channels. The discovery of AQP has provided insight into molecular mechanisms underlying renal water absorption and its regulation by vasopressin. Seven types of AQP have been identified in the kidney. AQP1 has been localized in the proximal tubule and descending thin limb, while AQP2, AQP3, and AQP4 are expressed in the collecting duct. Of these isoforms, AQP2 expression and intracellular trafficking is tightly regulated by vasopressin. Decreased expression of renal AQP has been detected in several disorders associated with polyuria and impaired ability to concentrate urine, as exemplified by nephrogenic diabetes insipidus or renal failure. In contrast, increased expression of AQP is seen in conditions leading to water retention, such as congestive heart failure, liver cirrhosis, and syndrome of inappropriate antidiuretic hormone secretion. Thus, the understanding of molecular structure and function of aquaporins may have important implications for therapy of water balance disorders.

Aquaporins↗
PubMed2001

Prolonged exposure to glucose degradation products impairs viability and function of human peritoneal mesothelial cells.

Bioincompatibility of peritoneal dialysis fluids (PDF) has been linked to the presence of glucose degradation products (GDP). Previous experiments have shown that short-term exposure to several GDP at concentrations found in commercially available PDF had no significant effect on human peritoneal mesothelial cells (HPMC). During continuous ambulatory peritoneal dialysis, however, cells are continually exposed to GDP for extended periods of time. Thus, the impact of GDP on HPMC during long-term exposure was assessed. HPMC were cultured for up to 36 d in the presence of 6 identified GDP (acetaldehyde, formaldehyde, furaldehyde, glyoxal, methylglyoxal, and 5-HMF) at doses that reflect their concentrations in conventional PDF. At regular time intervals, the ability of HPMC to secrete cytokines (interleukin-6 [IL-6]) and extracellular matrix molecules (fibronectin) was evaluated. In addition, cell viability, morphology, and proliferative potential were assessed. Exposure to GDP resulted in a significant reduction in mesothelial IL-6 and fibronectin release. Approximately 80% of this decrease occurred during the first 12 d of the exposure and was paralleled by a gradual loss of cell viability and development of morphologic alterations. After 36 d of exposure, the number of cells in GDP-treated cultures was reduced by nearly 60%. However, GDP-treated cells were able to resume normal proliferation when transferred to a normal GDP-free medium. HPMC viability and function may be impaired during long-term exposure to clinically relevant concentrations of GDP, which suggests a potential role of GDP in the pathogenesis of peritoneal membrane dysfunction during chronic peritoneal dialysis.

Aldehydes↗
PubMed2000

Effect of glucose degradation products on human peritoneal mesothelial cell function.

Bioincompatibility of conventional glucose-based peritoneal dialysis fluids (PDF) has been partially attributed to the presence of glucose degradation products (GDP) generated during heat sterilization of PDF. Most previous studies on GDP toxicity were performed on animal and/or transformed cell lines, and the impact of GDP on peritoneal cells remains obscure. The short-term effects of six identified GDP on human peritoneal mesothelial cell (HPMC) functions were examined in comparison to murine L929 fibroblasts. Exposure of HPMC to acetaldehyde, formaldehyde, glyoxal, methylglyoxal, furaldehyde, but not to 5-hydroxymethyl-furfural, resulted in dose-dependent inhibition of cell growth, viability, and interleukin-1beta (IL-1beta)-stimulated IL-6 release; for several GDP, this suppression was significantly greater compared with L929 cells. Although the addition of GDP to culture medium at concentrations found in PDF had no major impact on HPMC function, the exposure of HPMC to filter-sterilized PDF led to a significantly smaller suppression of HPMC proliferation compared to that induced by heat-sterilized PDF. The growth inhibition mediated by filter-sterilized PDF could be increased after the addition of clinically relevant doses of GDP. These effects were equally evident in L929 cells. In conclusion, GDP reveal a significant cytotoxic potential toward HPMC that may be underestimated in test systems using L929 cells. GDP-related toxicity appears to be particularly evident in experimental systems using proliferating cells and the milieu of dialysis fluids. Thus, these observations may bear biologic relevance in vivo where HPMC are repeatedly exposed to GDP-containing PDF for extended periods of time.

Cell Division↗
PubMedDate not supplied

Mesothelial toxicity of peritoneal dialysis fluids is related primarily to glucose degradation products, not to glucose per se.

OBJECTIVES: High concentrations of glucose and/or formation of glucose degradation products (GDPs) during heat sterilization of peritoneal dialysis fluids (PDFs) are believed to be key factors in the limited biocompatibility of PDFs. We have previously shown that several identified GDPs can specifically impair human peritoneal mesothelial cell (HPMC) function. In the present study we aimed at differentiating the respective roles of glucose and GDPs in the toxicity of PDF to mesothelial cells. METHODS: HPMCs were acutely pre-exposed to or incubated chronically in the presence of pH-neutral PDF sterilized by either heat (H-PDF) or filtration (F-PDF). In addition, HPMCs were treated with commercially available H-PDF manufactured either conventionally, that is, in single-chamber containers, or using novel dual-chamber bags that help to substantially decrease GDP formation. Functional assessment of HPMCs included viability, release of interleukin (IL)-6, and proliferation. RESULTS: Viability and release of IL-6 from HPMCs pretreated with H-PDF (pH 7.3) for 1 to 4 hours were significantly reduced compared to cells exposed to corresponding F-PDF. Incubation in medium mixed (1:1) with H-PDF considerably impaired growth of HPMCs, and over a period of 10 days gradually decreased both the viability of HPMCs and their ability to generate IL-6. These effects were either absent from or significantly less in HPMCs exposed to F-PDF. Similar differences were observed when commercial GDP-containing H-PDFs were compared with newly designed H-PDFs free of GDPs. CONCLUSIONS: Impaired viability and function of HPMCs exposed to glucose-containing pH-neutral PDF is related predominantly to the presence of GDP and, to a significantly lesser extent, to the presence of glucose per se. Prevention of GDP formation during autoclaving markedly improves the biocompatibility of H-PDF with HPMCs.

Cell Division↗
PubMedDate not supplied

Evidence for less irritation to the peritoneal membrane in rats dialyzed with solutions low in glucose degradation products.

BACKGROUND: Acidic pH and the presence of glucose degradation products (GDP) are believed to compromise the biocompatibility of peritoneal dialysis fluids (PDF). The present study examines the effects of long-term exposure to GDP and low pH by comparing conventional PDF and a new, neutral pH, low GDP solution. METHODS: All experiments were performed using a chronic infusion model of dialysis in nonuremic rats. The animals were treated for 6 weeks with 2 daily injections of 4.25% glucose-containing PDF. The following PDF were tested: CAPD3 (single-chamber bag, low pH, high GDP), CAPD3 pH 7.4 (single-chamber bag, neutral pH, high GDP), CAPD3-Balance (double-chamber bag, neutral pH, low GDP). All test solutions were obtained from Fresenius Medical Care, Bad Homburg, Germany. RESULTS: After 6 weeks of exposure, peritoneal permeability to water, urea, creatinine, glucose, and sodium, assessed by peritoneal equilibration test, was similar in all groups. However, compared to other PDF, dialysis with CAPD3-Balance was associated with reduced concentrations of protein and hyaluronan in the dialysate, decreased peritoneal eosinophilia, and reduced dialysate levels of chemokines CCL2/MCP-1 and CCL5/RANTES. Morphologic changes in the peritoneal membrane of CAPD3-Balance-treated animals were much less pronounced and included reduced vascular density, preservation of the mesothelial monolayer and intercellular junction, and no reduplication of the submesothelial basement membrane. CONCLUSIONS: A new generation of PDF with physiologic pH and low GDP level produce less irritation to the peritoneal membrane and better preserve its structural integrity. This effect seems to be related predominantly to minimized GDP concentrations.

Animals↗
PubMedDate not supplied

The role of the glyoxalase pathway in reducing mesothelial toxicity of glucose degradation products.

BACKGROUND: The glucose degradation products (GDP) presentin conventional peritoneal dialysis fluids (PDF) may exert adverse effects toward human peritoneal mesothelial cells (HPMC). Some GDP can be detoxified by the glyoxalase/ glutathione pathway. It has been shown that the addition of glyoxalase I (GLO-I) and reduced glutathione (GSH) to PDF effectively eliminates GDP. We have therefore examined the GLO-I/GSH system in HPMC and assessed the impact of GLO-I/ GSH-treated PDF on the viability and function of HPMC. METHODS: Heat-sterilized PDF (H-PDF) was incubated in the presence or absence of GLO-I and GSH for 1 hour at 37 degrees C, and then mixed with an equal volume of serum-free M199 medium and applied to HPMCin culture. After 24 hours, HPMC were assessed for viability, the release of interleukin-6, GLO-I activity, and cellular glutathione. The effects were compared to those exerted by filter-sterilized PDF (F-PDF), which was devoid of GDP. RESULTS: Exposure of HPMC to H-PDF resulted in reduced GLO-I activity, GSH depletion, and a decrease in cell viability. Pretreatment of H-PDF with either a combination of GLO-I and GSH or GSH alone markedly reduced inhibitory effects of H-PDF toward HPMC, as measured by cell viability and inter-Leukin-6 generation. Exposure of HPMC to the GSH precursor L-2-oxothiazolidine-carboxylic acid increased cellular GSH and prevented the loss of GLO-I activity in response to H-PDF. CONCLUSIONS: Exposure to conventional GDP-rich PDF impairs the activity of the glyoxalase/glutathione system in HPMC. Pretreatment of PDF with GSH or replenishment of cellular GSH protects HPMC against GDP-mediated toxicity.

Cells, Cultured↗