The multidirectional assessment of moderate caloric restriction and metformin treatment in obese patients.

Obesity treatment is often burdensome for patients. We used the combination of moderate caloric restriction (CR) with hypoglycemic metformin to assess their multidirectional effect in obese patients. One group was treated only with moderate CR (n=21) the second was treated with moderate CR and 800 mg metformin twice daily (n=23). Serum was drawn before and after treatment. The following parameters were monitored: anthropometric, cardiovascular, inflammatory, metabolic, and markers characteristic for thyroid, liver, pancreas, and kidney functions. Both tested groups did not significantly differ in most tested parameters after the treatment. Two groups reduced anthropometric parameters (body mass, body mass index (BMI), waist circumference) and fat mass but also muscle and fat-free mass, improving systolic blood pressure, insulin and leptin concentration, insulin sensitivity, leptin to adiponectin ratio, and inflammatory markers. Unfortunately, there was little impact on improving dyslipidemia and the thyroid and liver parameters. Free triiodothyronine (fT3) and gamma glutamyl transferase (GGT) activity were decreased in both groups, but triglycerides were reduced only in patients treated with moderate CR. Metformin with CR treatment decreases uric acid and aspartate aminotransferase (AspAT) activity. Metformin treatment with moderate CR in obese patients mainly improved insulin sensitivity, resulting in a reduction of patients with glucose intolerance, improved anthropometric, cardiovascular, and inflammatory mediators, and only slightly enhanced liver and thyroid function. No changes in kidney and pancreas function were observed during the treatment. In conclusion, eight weeks of CR alone and CR with metformin in obese adults improved anthropometric and metabolic markers, reduced muscle mass, fT3, GGT, proinflammatory, and CV parameters, and displayed no changes in kidney and pancreas function. The group treated with metformin after the treatment was still more obese and had higher C-reactive protein (CRP) and homeostasis model assessment-an index of insulin resistance (HOMA-IR), but despite this, considerably reduced the number of patients with glucose intolerance.

Soluble receptor for advanced glycation end products (sRAGE) correlates with obesity-related parameters, and it is not easy to be modified by moderate caloric restriction in obese humans.

Deficiency of a soluble form of the advanced glycation end products receptor (sRAGE) is implicated in obesity-induced complications. Serum sRAGE is inclined to be modified by changes in body weight. We analysed serum sRAGE concentrations in patients with obesity undergoing moderate calorie restriction, which mimics the real-life situation and is not harmful to obese humans. Serum sRAGE was measured by immunoassay in 50 patients with obesity who underwent calorie restriction by 300-500 kcal/day for 8 weeks. In effect calorie restriction resulted in an expected decrease in body weight (by 2.1 kg for an 8-week intervention, p<0.0001), as well as reduced systolic blood pressure, modified dyslipidemia (cholesterol, triglycerides), reduced obesity-related inflammation (tumor necrosis factor-alfa, interleukin-6, C-reactive protein), improved insulin sensitivity. However, it was not accompanied by any significant change in sRAGE concentration. There was a strong negative correlation between BMI and the sRAGE level. Accordingly, the levels of sRAGE were the highest in lean control. In conclusion: a modest weight reduction is unlikely to improve decreased sRAGE levels.

The role of purinergic P2Y12 receptor blockers on the angiogenic properties of endothelial cells: an in vitro study.

Pharmacotherapy with agents that inhibit platelet function has proven to be effective in the treatment of acute coronary syndrome. Proper re-endothelization after angioplasty prevents adverse cardiovascular events. Therefore, in this in vitro study we examined how antiplatelet P2Y12 receptor blockers can affect endothelial cells' angiogenic properties. Endothelial cells were exposed to ticagrelor, prasugrel and clopidogrel in their highest concentrations obtained in serum after the treatment with loading and clinical doses. Further, the viability, apoptosis, and necrosis were tested and the following angiogenic properties such as proliferation, migration, invasiveness, tube formation, wound healing and the production of angiogenic mediators (bFGF, PDGF, MMP-2, Ang-2, TIMP-1). The results of this study showed that P2Y12 receptor blockers in the tested concentrations are safe for endothelial cells. They neither induced necrosis or apoptosis nor changed the endothelial cell viability, migration, invasiveness, tube formation, wound healing, the production of VEGF or its receptors. However, they reduced cell proliferation. It was shown that out of these three drugs, ticagrelor in its loading concentration had the most potent angiogenic property. It reduced cell proliferation and changed the production of angiogenic (bFGF, MMP-2) and angiostatic mediators (Ang-2). In conclusion, P2Y12 receptor blockers in the concentrations obtained in the serum during standard therapy reduced endothelial cell proliferation. Despite this slight antimitogenic effect, they did not change endothelial cell tube formation or wound healing. Out of the three tested drugs, ticagrelor had the most potent angiogenic effect in vitro, but not strong enough to disturb tube formation and wound healing.

Weight loss-dependent and -independent effects of moderate calorie restriction on endothelial cell markers in obesity.

Endothelial cell dysfunction in obesity can be reduced by calorie restriction (CR), however it is unclear whether this benefit requires a concomitant weight loss or is it simply related to the reduced calorie intake per se. In our study serum was drawn from 41 obese women who were undergoing an 8-week dietary intervention with 15 - 30% energy deficit, and from 48 age- and sex-matched controls of normal weight. Serum was analysed for biomarkers of endothelial cell function, oxidative stress and inflammation. Compared with non-obese individuals, the obese patients had lower serum levels of nitric oxide (NO), adiponectin, and decreased serum antioxidant status. They also had significantly higher levels of adhesive molecules, thrombomodulin (TM), von Wilebrand factor (vWF), asymmetric dimethylarginine (ADMA), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and leptin. To further characterize the effect of moderate CR, the patients were ranked into two comparable groups according to the extent of weight loss - below and above the median (-5.8 kg). A moderate dietary intervention did not correct adiponectin, antioxidant status, vWF, TM, and plasminogen activator inhibitor-1 (PAI-1) but ameliorated changes in other parameters. Only changes in NO and - to a lesser degree - in sE-selectin showed a clear relationship with the magnitude of weight reduction. By contrast, a beneficial reduction in TNF-α occurred equally in patients who lost more or less weight after caloric restriction. We concluded that moderate calorie restriction could still improve several parameters of endothelial cell function irrespective of whether it was accompanied by changes in body mass. However, a significant improvement in nitric oxide, a key mediator of endothelial well-being, requires a substantial reduction in body weight.

The role of mTOR inhibitors and HMG-CoA reductase inhibitors on young and old endothelial cell functions, critical for re-endothelialisation after percutaneous coronary intervention: an in vitro study.

Percutaneous coronary intervention (PCI) has become a standard treatment in patients with acute coronary syndrome. However, it is associated with endothelial cell denudation, which may predispose to in-stent thrombosis and restenosis. Pharmacological methods which prevent restenosis can delay post-PCI re-endothelialisation. We have therefore examined how atorvastatin (HMG-CoA reductase inhibitor), sirolimus and everolimus (mTOR inhibitors) affect young and old endothelial cell functions which are responsible for wound healing after PCI. Replicative senescence was induced by serial passages of human umbilical vein endothelial cells (HUVECs). The cells which were examined at their first passages and last passages were designated as 'young' and 'old' respectively. Young and old endothelium were grown to confluence and were wounded by scraping. Scratch healing in the presence or absence of atorvastatin (AT), rapamycin (SR) and everolimus (EV) was monitored by time-lapse microscopy. In addition cells were assessed for viability (MTT assay), migration (chemotaxis chamber), proliferation (3H-thymidine), and cytokine production (immunoassays). Senescent endothelial cells produce more proinflammatory cytokines, angiogenic VEGF and extracellular matrix proteins. They stop proliferating and have diminished migration. When compared to young endothelium, they have similar viability and can regenerate wounds in comparable time. The drugs that have been tested have anti-inflammatory properties but even after pretreatment old cells still produced significantly higher concentration of tested mediators in comparison with young ones. In the concentration obtained in serum after stent implantation, mTOR inhibitors in dose-dependent manner reduced cell proliferation, migration and wound healing. Reduced healing is more pronounced in young endothelium. Atorvastatin, at clinically relevant concentration, is safe for young and old cells. Atorvastatin, sirolimus and everolimus inhibited the secretion of pro-inflammatory mediators in young and old endothelium. In concentrations seen in serum during standard therapy, rapalogs impair endothelial cell regeneration after injuries mimicking those occurring during PCI, while atorvastatin does not affect the healing.

Simple obesity and renal function.

Increasing evidence accumulate to suggest that obesity increases the risk of chronic kidney disease independently of dyslipidemia, diabetes, and hypertension. This so-called obesity-related glomerulopathy is characterized at early stages by glomerular hypertrophy with or without secondary focal segmental glomerulosclerosis. Since, however, kidney biopsies are usually not performed at this phase, an early diagnosis of the disease is often difficult. Here, we review new developments in the pathophysiology of obesity-associated kidney dysfunction and discuss the potential of appropriate monitoring of glomerular filtration rate and albuminuria for early detection of the disease. We also present the benefits conferred by even moderate dietary restriction on the course of the disease.

Atorvastatin does not impair endothelial cell wound healing in an in vitro model of vascular injury.

Percutaneous coronary intervention (PCI) became standard treatment modality for coronary revascularization. However, it is associated with endothelial cell denudation, which may predispose to in-stent thrombosis and restenosis. Since statins may inhibit vascular cell proliferation, one may fear that their early administration will delay post-PCI re-endothelialization. We have therefore employed an in vitro scratch assay to examine how atorvastatin affects endothelial cell wound healing. Human umbilical vein endothelial cells (HUVEC) were grown to confluence and wounded by scraping. Scratch healing in the presence or absence of atorvastatin was monitored by time-lapse photo-microscopy. In addition cells were assessed for viability (MTT assay), migration (chemotaxis chamber), proliferation ((3)H-thymidine and bromodeoxyuridine incorporation), and cytokine production (immunoassays). The exposure of HUVEC to atorvastatin resulted in a dose-dependent decrease in cell viability and proliferation. However, this effect was observed only at doses ≥1 µM, which is well above the concentrations seen in vivo. At clinically relevant doses (≤0.1 µM) atorvastatin did not impair wound closure, nor did it inhibit cell viability, proliferation, and migration. It did however reduce the constitutive and the stimulated release of cytokines (IL-6, IL-8, MCP-1), adhesion molecules (sICAM-1) and matrix proteins (fibronectin). We conclude that atorvastatin at doses corresponding to concentrations seen in serum during standard therapy does not impair endothelial cell regeneration after injuries mimicking those occurring during PCI. It does, however, inhibits the secretion of pro-inflammatory mediators.

Role of mesothelial cell-derived granulocyte colony-stimulating factor in interleukin-17-induced neutrophil accumulation in the peritoneum.

Recent studies suggest that peritoneal CD4(+) T lymphocytes may control recruitment of polymorphonuclear leukocytes (PMN) during peritonitis by an interleukin-17 (IL-17)-dependent mechanism. IL-17 and granulocyte colony-stimulating factor (G-CSF) have been proposed to form an axis that regulates PMN transmigration. Here we report on the role of G-CSF released by human peritoneal mesothelial cells (HPMCs) in IL-17A-mediated peritoneal PMN accumulation. In vitro exposure of HPMCs to IL-17A resulted in a time- and dose-dependent release of G-CSF. This effect was related to the induction of G-CSF mRNA and mediated through the nuclear factor-kappaB (NF-kappaB) pathway. The novel observation was that IL-17A-stimulated NF-kappaB activation in HPMCs followed a biphasic profile, with an early induction (45 min), followed by the return to basal levels (90 min), and a delayed induction (3 h). Tumor necrosis factor alpha synergistically amplified IL-17A-induced G-CSF production by enhanced NF-kappaB activation and through stabilization of G-CSF mRNA. Intraperitoneal (i.p.) administration of IL-17A in Balb/c mice resulted in increased local levels of G-CSF and selective PMN accumulation. Administration of anti-G-CSF blocking antibody before IL-17A injection significantly reduced the IL-17A-triggered PMN infiltration. This effect occurred despite increased i.p. levels of PMN-specific chemokines KC and macrophage inflammatory protein-2 seen in animals treated with anti-G-CSF antibody. These data demonstrate that the mesothelium-derived G-CSF plays an important role in IL-17A-induced PMN recruitment into the peritoneum.

Plasma glutathione peroxidase activity in the elderly.

Schiavon et al. (1994) have reported that the measurement of plasma glutathione peroxidase activity (PGP) could provide an index of renal function. Its activity, which was depressed in patients with impaired renal function, correlated positively with creatinine clearance and negatively with serum creatinine. To evaluate the hypothesis that the plasma PGP activity may be used to assess renal function in elderly, we measured the plasma PGP and creatinine clearance (ClCr) in 65 active, community-dwelling elderly (range: 65-93 years; 47 women and 18 men). We did not include persons with advanced renal failure in our study. PGP did not correlate with PCr and it was similar among patients with normal and with increased PCr (127.0 +/- 30.7 U/l and 119.7 +/- 21.6 U/l, respectively). A positive correlation was found between PGP and ClCr (r = 0.30; p < 0.01). Plasma PGP activity was lower in patients with a ClCr lower than 70 ml/min/1.73 m2 than in those who had a higher ClCr (113.0 +/- 25.8 U/l and 131.2 +/- 26.7 U/l, p < 0.01). However, no correlation was found between ClCr and PGP in subjects with lower ClCr. PGP did not correlate with age but there was a correlation between ClCr and age (r = -0.24, p < 0.05). Our results suggest that plasma PGP activity is decreased in the patients with impaired renal function but this decrease does not correlate with age-dependent decline of kidney function.

The effect of a nitric oxide inhibitor (L-NAME) on peritoneal transport during dialysis in rats.

OBJECTIVE: To assess the effect of an inhibitor of nitric oxide synthesis [N(G)-nitro-L-arginine methyl ester (L-NAME)] on peritoneal transport during peritoneal dialysis (PD) and peritonitis in rats. METHODS: The authors studied peritoneal transport of small and large solutes, and net ultrafiltration (UF) in rats during PD with Dianeal 3.86 (Baxter, McGaw Park, IL, U.S.A.). They evaluated the effect of L-NAME used as an additive to dialysis fluid in concentrations 0.5-5 mg/mL on peritoneal transport of small and large molecules and on transperitoneal UF. In addition, they studied the effect of L-NAME (5 mg/mL) during acute peritonitis induced by lipopolysaccharides (5 microg/mL) given intraperitoneally. RESULTS: The addition of L-NAME to dialysis fluid increased the selectivity of the peritoneum and net UF during dialysis. Lipopolysaccharides used as an additive to the dialysis fluid, together with L-NAME, did not induce changes in transperitoneal transport of small and large solutes and did not cause a significant decline in net UF. L-NAME given intraperitoneally reduced both local and systemic production of nitric oxide, which might explain its effects on peritoneal transport. CONCLUSIONS: Nitric oxide is an important mediator of changes in peritoneal transport and its effect is especially significant during peritonitis.

N-acetylglucosamine changes permeability of peritoneum during chronic peritoneal dialysis in rats.

OBJECTIVE: To evaluate the effect of supplementation of dialysis fluid with N-acetylglucosamine (NAG) on the permeability of peritoneum during chronic peritoneal dialysis in rats. DESIGN: Experiments were performed on rats with surgically implanted peritoneal catheters. Dialysis solution [Dianeal 1.5% (Baxter, Deerfield, IL, U.S.A.) supplemented with either NAG 50 mmol/L or glucose 50 mmol/L (control)] was infused intraperitoneally twice, every day, for 8 weeks. Peritoneal equilibration tests (PET) were performed in all animals at the beginning of the study and after 8 weeks of dialysis. Additionally, at the end of each week, dialysis solution infused in the morning was drained after 4 hours of intraperitoneal dwell. White blood cell count, creatinine, and total protein concentrations were measured in the effluent dialysate. After 8 weeks of dialysis, the morphology of the peritoneum was studied. RESULTS: In rats exposed to dialysis fluid supplemented with NAG, peritoneal permeability to creatinine and proteins was reduced when compared to animals dialyzed with glucose solution. In NAG treated animals, staining with alcian blue for polyanions in the peritoneal interstitium was significantly stronger than in rats dialyzed with glucose solution. CONCLUSIONS: Chronic peritoneal dialysis with dialysis solution supplemented with N-acetylglucosamine causes accumulation of glycosaminoglycans in the peritoneal interstitium, which results in a change of peritoneal permeability.

Peritoneal surface area and its permeability in rats.

OBJECTIVE: Evaluation of peritoneal surface area and its permeability during dialysis in rats of various ages. DESIGN: Study I: planimetry of peritoneum and its topographic areas was performed in 47 rats of various ages (8-30 weeks). Study II: net ultrafiltration (UF), dialysate-to-serum ratios for urea, creatinine, albumin, and total protein as well as their peritoneal permeability coefficients, were measured during a 1-hour peritoneal exchange with Dianeal 2.5%, in 21 rats of different ages (9-30 weeks) and with various peritoneal surface areas. ANIMALS: Male Wistar rats. RESULTS: The peritoneal surface area in rats increases during aging, but young animals with lower body weight have a relatively larger peritoneal surface area than older, larger animals. The area of the topographic fragments of peritoneum expressed as a percentage of the total peritoneal surface is steady during aging. Efficiency of transperitoneal water removal expressed as net UF per amount of absorbed glucose declines in older animals, with larger peritoneal surface areas. Dialysate/serum ratio of solutes transported from blood to dialysate is proportional to peritoneal surface area. Permeability coefficient (K) of peritoneum to urea and creatinine is unchanged during the aging of animals. However peritoneal permeability (K) to albumin increases during aging, with the opposite tendency for total proteins. CONCLUSIONS: Kinetics of peritoneal dialysis in rats of different ages is determined by peritoneal surface area and permeability of peritoneum to individual solutes.

Long-term effects of glycylglycine peritoneal dialysis solution with neutral pH on peritoneum in rats.

This study was designed to test the morphological and functional effects of neutral, bicarbonate-based peritoneal dialysis solution containing glycylglycine on the peritoneum of chronically dialyzed rats. Peritoneal dialysis catheters were implanted in 36 rats. The animals were dialyzed twice daily for 4 weeks with a solution containing bicarbonate (35 mmol/L), glycylglycine (10 mmol/L), and 4% of anhydrous glucose (pH 7.35) (group 1; n = 18) or with lactate-based standard 4.25% Dianeal (pH 5.3 (group 2; n = 18). At the beginning of the study, reabsorption of glucose was slower in group 1 (p < 0.02); at the same time, the hyaluronic acid level in the effluent was higher in this group (p < 0.05). However, towards the end of the study these differences disappeared. After 4 weeks of dialysis in rats exposed to bicarbonate-based solution only, the transperitoneal loss of proteins was slower. In morphological studies of the parietal peritoneum, we detected no statistically significant differences between control nondialyzed rats and those exposed to tested solutions. In a biopsy of visceral peritoneum a tendency was observed for increased thickness of peritoneum in rats dialyzed with both tested peritoneal dialysis solutions when compared to control animals. In conclusion, neutral pH glycylglycine peritoneal dialysis solutions seem to be more biocompatible than standard dialysis solutions.

Alterations of intraperitoneal inflammation by the addition of L-2-oxothiazolidine-carboxylate.

The authors studied the effect of L-2-oxothiazolidine-carboxylate (OTZ), a substrate for intracellular glutathione synthesis, in an in vivo model of lipopolysaccharide (LPS)-induced peritonitis in rats. The addition of LPS to dialysis fluid increased the white blood cell (WBC) count and the nitrite (index of NO synthesis) level in the dialysate. The simultaneous addition of OTZ to the dialysis fluid prevented an increase of WBCs but not of nitrites in the dialysate. Intraperitoneal inflammation was accompanied by a decrease in net transperitoneal ultrafiltration, an increase in the absorption of glucose, and a loss of protein into the dialysate. OTZ partially reversed the effect of peritonitis on net ultrafiltration. Peritoneal leukocytes from rats exposed to LPS showed a reduced concentration of glutathione, an effect that was reversed in the presence of OTZ. These results show that the supplementation of dialysis fluid with OTZ modified the peritoneal reaction to acute inflammation.

In vivo model to study the biocompatibility of peritoneal dialysis solutions.

This study was designed to analyze the complex morphologic and functional effects of dialysis solutions on peritoneum in a rat model on chronic peritoneal dialysis. Peritoneal catheters were inserted into 10 male, Wistar rats and the animals were dialyzed twice daily for 4 weeks with 4.25% Dianeal. During the study we observed two opposite effects: healing of the peritoneum after catheter implantation--decreased cell count in dialysate, decreased permeability of the peritoneum to glucose and total protein, increased volume of drained dialysate; and damage to the membrane due to its exposure to peritoneal dialysis solution--increased hyaluronic acid levels in dialysate, a tendency of the peritoneum to thicken when compared to non-dialyzed animals. Our rat model of CAPD may be used for quantitative and qualitative assessment of the effects of peritoneal dialysis solution on the peritoneum during chronic dialysis.

Synthesis of hyaluronic acid by human peritoneal mesothelial cells: effect of cytokines and dialysate.

OBJECTIVE: To assess effects of the inflammatory cytokines (IL-1-beta, TNF-alpha, TGF-beta 1) and dialysate effluent on synthesis of hyaluronic acid by human peritoneal mesothelial cells (HMC) in in vitro culture. METHODS: Dialysate effluent was collected after the overnight dwell of Dianeal 1.5% from patients during CAPD training. HMC were obtained from omentum from nonuremic donors or were harvested from the dialysate effluent from CAPD patients. Synthesis of hyaluronic acid was studied on monolayers of HMC, which were deprived of serum 48 hours prior to experiment. Effects of cytokines were tested in a medium with low serum concentration (0.1%) or in medium mixed (1:1 v/v) with the autologous dialysate. Hyaluronic acid level in medium was measured with radioimmunoassay. RESULTS: Cytokines enhanced synthesis of hyaluronic acid by HMC, and the strongest effect was induced by IL-1. Effluent dialysate stimulates synthesis of hyaluronic acid stronger than 10% FCS. Effluent dialysate and IL-1 synergistically enhance synthesis of hyaluronic acid by HMC. CONCLUSION: Effluent dialysate from CAPD patients stimulates production of hyaluronic acid by HMC and acts synergistically with cytokines.

In vitro detection of hydrogen peroxide in mesothelial cells.

Hydrogen peroxide can be indirectly detected in mesothelial cells by measuring the rate of inhibition of the intracellular catalase activity by 3-amino-1, 2,3-triazole (AT). AT binds and inhibits catalase only in the presence of hydrogen peroxide, and the effect is proportional to the amount of hydrogen peroxide. The effect of AT on catalase activity is prevented in the presence of ethanol. In mesothelial cells in vitro exposed to dialysis fluids (Dianeal 1.36%, 2.27%, and 3.86%) no significantly increased generation of hydrogen peroxide was detected. However, interleukin-1 (IL-1) (10 ng/mL) enhances, within two hours (+40%, p < 0.05), intracellular production of hydrogen peroxide. The presented method of detection of intracellular hydrogen peroxide may be helpful in studies of the pathomechanisms causing mesothelial damage in conditions of peritoneal dialysis.

Functional properties of mesothelial cells after prolonged exposure to dialysate effluent.

The authors tested in vitro the effect of glucose-based and amino acid-based dialysate effluent on the function of human peritoneal mesothelial cells. After 9 days of exposure to the tested effluents with medium (1:1 v/v) or to a medium supplemented with 10% fetal calf serum (FCS) (control), several functional properties of the cells were studied. The synthesis of DNA measured by incorporation of 3H-methyl-thymidine was higher in mesothelial-cell monolayers exposed to the dialysates than in the controls. Synthesis of hyaluronic acid was similar in all three groups, but after stimulation with Il-1 the cells exposed to the dialysates produced more hyaluronic acid. Synthesis of tissue plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) was higher in the control cells. However, after stimulation with IL-1, the cells exposed to the dialysate showed greater synthesis of PAI-1 than of t-PA. Also, procoagulant activity of the control cells was higher than that of the cells exposed to the dialysates. We have concluded that the functional properties of the mesothelial cells may be altered in vitro during prolonged exposure to the dialysate, something that may also occur in vivo.