Pathophysiology of ageing.

Ageing is characterized by a gradual decline in organ functional reserves which reduces the ability to maintain homeostasis under conditions of stress. Introduction of cell culture and molecular biology techniques has provided new experimental tools for the analysis of ageing at the molecular level. During ageing progressive degeneration of cells and loss of regenerative capacity are enhanced and with time the alterations caused by them ultimately lead to death. In this paper the current knowledge of the mechanisms of ageing is summarized.

Insulin stimulates the activity of Na+/K(+)-ATPase in human peritoneal mesothelial cells.

OBJECTIVE: To assess the effect of insulin on the Na+/K(+)-ATPase expression and activity in human peritoneal mesothelial cells (HPMC). METHODS: HPMC were isolated from the omental tissue of non-uremic patients, grown to confluence and rendered quiescent by serum deprivation for 24 hours. The activity of Na+/K(+)-ATPase was determined by measuring the ouabain-sensitive 86Rb uptake. To assess whether the effect of insulin was related to changes in [Na+]i the sodium influx was measured with 22Na and the activity of Na+/K(+)-ATPase was assessed in the presence of amiloride. Expression of Na+/K(+)-ATPase alpha 1,alpha 2 and beta 1-subunit mRNAs was determined by RT/PCR. RESULTS: Exposure of HPMC to insulin resulted in a time- and dose-dependent increase in the Na+/K(+)-ATPase activity. After 60 minutes the ouabain-sensitive 86Rb uptake (cpm/10(4) cells) was increased from 6650 +/- 796 in control cells to 9763 +/- 1212 in HPMC exposed to 100 mU/mL insulin (1.5-fold increase; n = 4, P < 0.05). In addition, incubation of HPMC with 100 mU/mL insulin resulted in a time-dependent increase in the 22Na influx. Pre-exposure of HPMC to 1mM amiloride reduced the activity of Na+/K(+)-ATPase but did not block the stimulatory effect of insulin. RT/PCR analysis revealed that HPMC constitutively expressed alpha 1- and beta 1-subunit mRNAs while the alpha 2-subunit mRNA was barely detectable. Exposure of HPMC to insulin for up to 24 hours was not associated with any changes in the expression of either alpha 1, alpha 2 or beta 1-subunit. CONCLUSION: Insulin stimulates the Na+/K(+)-ATPase activity in HPMC in a time- and dose-dependent manner. This effect appears to mediated by an increase in [Na+]i and is not related to alterations in Na+/K(+)-ATPase subunit mRNAs expression.

A method for measurements of hydraulic conductivity of biological membranes in vitro.

The results of measurements of water flow across four isolated epithelial membranes (skin of frogs, colon of rats, parietal peritoneum and mesentery of rabbits) with hydrostatic pressure gradient as volume driving force have been presented herein. The membranes were mounted between two semichambers filled with Ringer or Hanks solution; the volume on both sides of the membrane was strictly controlled by photogauges coupled with microdosimeters. Transmembrane volume flow (Jv; nL x sec-1 x cm-2) induced by the hydrostatic pressure gradient (delta P; atm) was measured with the use of an electronic device connected with the photogauges, and with a computer which monitored values throughout the experiment. Hydraulic conductivity index (Lp) was calculated from the monitored values (Lp = Jv/delta P; in cm x sec-1 x atm-1). The index proved to be a characteristic property of each studied membrane. Effects of some factors thought to influence the permeability of the membranes were checked: Hypertonicity of medium bathing the frog skin and parietal peritoneum induced an increase of permeability, whereas addition of mercuric chloride diminished it in the frog skin, the mesentery and the colon. The elaborated method can be useful in further studies on water metabolism and on participation of various barriers in water epithelial transport processes.

Species-dependent topography of the peritoneum.

Planimetric studies of peritoneal surface area were performed in 10 humans, 12 rabbits, and 15 rats. It was found that the total peritoneal surface area (TPSA) correlated in humans with body surface area (BSA) (r = 0.98, p < 0.0001) and body weight (r = 0.93, p < 0.001), and correlated in animals with body weight (r = 0.80, p < 0.005 in rabbits; and r = 0.88, p < 0.0001 in rats). The area of parietal peritoneum was 18.1 +/- 1.8% of TPSA in humans, 17.8 +/- 1.0% of TPSA in rabbits, and 22.6 +/- 2.1% of TPSA in rats (p < 0.001 vs humans and rabbits). Additionally, the area of peritoneum covering the individual organs (expressed as % of TPSA) was different in humans, rabbits, and rats: for example, the area of peritoneum covering the diaphragm was 6.4 +/- 1.5% of TPSA in humans, which was larger than in animals (3.0 +/- 0.3% in rats, p < 0.0001 vs humans and rabbits; and only 2.1 +/- 0.4% in rabbits, p < 0.0001 vs humans). Presented results show that interspecies variation in the topography of the peritoneum should be taken into account when the results from experimental studies done on animals are extrapolated to humans.

[Functional aspects of topographic differentiation in the peritoneum]. / Funkcjonalne aspekty zróznicowania topograficznego otrzewnej.

Volumetric measurements have been performed in vitro to define permeability for water of different parts of peritoneum. The experiments were carried out on isolated fragments of rabbit mesentery, anterior abdominal wall, and diaphragm; mesothelial layer lining these membranes was retained in one group, whereas in another one it was intentionally desquamated. The membranes were mounted between two semichambers filled with Hanks solution; the volume on both sides of the membrane was strictly controlled by photo-gauges coupled with microdosi-meters. Transmembrane volume flow (Jv; nL/sec cm2) induced by hydrostatic pressure gradient (delta P; atm) was measured using an electronic device connected with the photo-gauges, and joined with a computer which monitored the values throughout the experiment. Hydraulic conductivity (Lp; in cm/sec atm) and index of impediment (Zp; in sec atm/cm) were calculated from the monitored values. Significant differences between the three types of peritoneal membrane were observed: the hydraulic conductivity of diaphragm was lowest, and the impediment highest (0.86 +/- 0.11 x 10(-2) and 116.3 +/- 12.2, respectively) contrasting with the corresponding values of mesentery which were highest, and lowest (38.9 +/- 3.7 x 10(-2), and 2.57 +/- 0.25, resp.); peritoneum taken from anterior abdominal wall showed intermediate values (6.3 +/- 0.56 x 10(-2), and 15.9 +/- 1.5, resp.). Removal of mesothelium induced significant increase of the permeability of diaphragm and anterior abdominal wall, however, no such effect was observed in mesentery. The obtained results seem to show that various parts of peritoneum may function qualitatively different during dialysis, however, any conclusion concerning clinical aspects of the dialysis should be drawn with all proper reserves.

Effect of methotrexate, doxorubicin and mitoxantrone on human peritoneal mesothelial cell function in vitro.

Recent studies have demonstrated that intraperitoneal instillation of dialysis solutions and drug additives may adversely affect the function of peritoneal cell populations. Therefore the aim of the present investigation was to examine the short-term effects of antineoplastic agents on human peritoneal mesothelial cells (HPMC). We have assessed the integrity of HPMC membrane and mechanisms of intracellular potassium transport. There was no evidence of significant cytotoxicity (as measured by 86Rb release) during a 60-min exposure of HPMC to either methotrexate (10(-6)-10(-4) M), doxorubicin (10(-7)-10(-5) M) or mitoxantrone (10(-7)-10(-5) M). In HPMC exposed to doxorubicin (10(-6) M) the intracellular transport of potassium, as assessed with 86Rb as its analogue, was not affected. Methotrexate (10(-5) M) diminished Na,K-ATPase activity and simultaneously enhanced the 86Rb transport via furosemide-sensitive pathway. Mitoxantrone reduced the furosemide-sensitive 86Rb influx in a dose-dependent manner and at a concentration of 10(-4) M it also impaired the ouabain-dependent 86Rb influx. These data demonstrate that antineoplastic agents interfere with HPMC function which might contribute to the oncostatic-induced peritoneal toxicity.

[Transforming growth factor-beta: a new approach to the pathogenesis of glomerulonephritis and glomerulosclerosis]. / Transformujacy czynnik wzrostowy--beta: nowe spojrzenie na patogeneze zapalenia i stwardnienia klebków nerkowych.

TGF-beta is a multifunctional regulatory peptide which plays a key role in embryogenesis, tissue repair and immune response. One of the most important properties of TGF-beta is its widespread effects on extracellular matrix. TGF-beta stimulates the synthesis of individual matrix components and reduces the matrix degradation by proteases. It also modulates the expression of integrin receptors that mediate cell-matrix interactions. Moreover it has been demonstrated that TGF-beta may be involved in the pathogenesis of diseases characterized with an excessive matrix accumulation. In this review we summarize data from experimental studies which link the action of TGF-beta with the pathological matrix deposition in glomerulonephritis. We also present compelling evidence that the sustained expression of TGF-beta contributes to the development of glomerulosclerosis and progressive kidney fibrosis. Finally we discuss prospects of anti-TGF-beta therapy in treating renal diseases associated with overproduction of TGF-beta.

In vitro culture of human peritoneal mesothelium for investigation of mesothelial dysfunction during peritoneal dialysis.

There is a growing need for more biocompatible peritoneal dialysis fluid formulations. Using a method of in vitro cell culture we have examined the effect of osmotic agents, antiseptics and drug additives on various functions of human peritoneal mesothelial cells (HPMC). We have demonstrated that glucose and amino acids reduce the HPMC proliferation rate in a dose-dependent manner. 3H-methyl-thymidine incorporation into HPMC exposed to either 1.1% amino acid mixture or 2.0% glucose was decreased to (mean +/- SD) 32.5 +/- 6.1% and 16.8 +/- 1.2% of the control level respectively. Cultured HPMC have been shown to produce a fibrinolytic activity which may be estimated by the rate of plasma clot lysis. This activity may be diminished following the pre-incubation with 2.0% glucose and 1.1% amino acid mixture. We have found that povidone-iodine exerts a dose-dependent cytotoxic effect towards HPMC as measured by the increased 86Rb or 3H-inulin release from radiolabelled cells. We have also demonstrated that insulin enhances the 86Rb uptake and stimulates the Na, K-ATPase activity in HPMC but on the other hand is capable of reducing the phospholipids secretion from HPMC. Glucose, hydrocortisone or verapamil have also been shown to inhibit the release of mesothelial phospholipids. These data indicate that the mesothelial cell culture provides a convenient model for testing the biocompatibility of peritoneal dialysis solutions and assessing the dysfunction of mesothelial cells during peritoneal dialysis.

Glycerol toxicity for human peritoneal mesothelial cells in culture: comparison with glucose.

Glycerol has been proposed as a substitute osmotic agent for glucose in peritoneal dialysis fluids. We have compared the effect of glycerol and glucose on the function of human peritoneal mesothelial cells (HPMC) in vitro. The viability of HPMC was not affected by glycerol (up to 250 mM), whereas it was reduced by glucose in a time- and dose-dependent manner, as assessed by the LDH release. Although the incubation of HPMC with glycerol induced a dose-dependent decrease in HPMC proliferation, the effect was significantly less inhibitory than that produced by glucose. In HPMC treated with 90 mM of glycerol or glucose the incorporation of [3H]-thymidine had reached 79.0 +/- 19.3% and 55.3 +/- 4.0% of the control (p < 0.05 and p < 0.01), respectively. As measured by the [methyl-14C]-choline incorporation, the intracellular amount of newly synthesized phospholipids was reduced from (cpm/microgram cellular protein) 147 +/- 58 in control HPMC to 59 +/- 15 in cells exposed to 90 mM of glucose (p < 0.01), but not affected by glycerol (163 +/- 65). On the other hand, both glycerol and glucose (90 mM) decreased the synthesis of proteins (as assessed by the [3H]-proline incorporation) and interfered with potassium (86Rb) transport mechanisms in HPMC. Our data suggest that there exist some possibly advantageous aspects of glycerol as far as mesothelial cell biocompatibility profile is concerned.

Effect of phosphatidylcholine on the function of human mesothelial cells in vitro.

We tested the hypothesis that phosphatidylcholine (PC) molecules present in the dialysis solution may interact with the mesothelial cell membrane and modify its function. In vitro experiments were performed on human mesothelial cells (HMC) in culture. PC decreased proliferation of HMC when used at concentrations of 200 mg/l and higher. PC was also cytotoxic to HMC as measured by the release of lactate dehydrogenase from their cytosol. Cells exposed to PC had a diminished capacity for taking up 86Rb from medium. PC decreased the fibrinolytic properties of HMC and increased their procoagulant activity. Our results suggest that the positive short-term effect of the addition of PC to the dialysis solution (i.e., an increase in ultrafiltration) may be over-shadowed by its deleterious action on HMC membrane.

In vitro study of the mechanism of potassium transport into human mesothelial cells. I: Effect of hyperosmolality.

OBJECTIVE: To study the mechanism(s) of potassium transport into human mesothelial cells (HMC) exposed to osmotic solutes. DESIGN: Using potassium analog 86Rb, we evaluated its intracellular transport through three pathways: 1. blocked by ouabain; 2. blocked by furosemide but not by ouabain; 3. blocked by neither furosemide nor ouabain. Experiments were performed in a normotonic medium (control) or in a medium supplemented with osmotic solutes (glucose, glycerol, mannitol). Both the acute and chronic effects of osmotic solutes on potassium transport were studied. RESULTS: The acute exposure of mesothelial cells to osmotic solutes modifies the intracellular transport of potassium through all studied channels, and the effect is specific for every solute. In mesothelial cells exposed over 7 days to glucose (90 mM), the intracellular transport via ouabain- and furosemide-blocked channels is decreased, whereas it is increased through the third pathway. Total intracellular accumulation of 86Rb (potassium) ions in mesothelial cells cultured in a medium supplemented with various concentrations of glucose is decreased, and this effect is proportional to the concentration of glucose in the medium. CONCLUSIONS: The intracellular transport of potassium in mesothelial cells is regulated through at least three independent mechanisms. Acute or chronic exposure of mesothelial cells to a hypertonic medium affects the intracellular accumulation of potassium, and this effect is specific for the various osmotic solutes.

Chondroitin sulphate and peritoneal permeability.

We studied the effect of chronic intraperitoneal (ip) infusion of saline supplemented with the glycosaminoglycan-chondroitin sulphate 0.1% on the permeability and peroxidation of the peritoneal membrane in rats and compared this with the effect of saline infusion alone. Animals treated with chondroitin sulphate had a higher net ultrafiltration (uf), a slower glucose absorption from the dialysate and less trans-peritoneal loss of proteins. Chronic ip infusion of chondroitin sulphate reduced peroxidation of the peritoneum. These observations suggest that chondroitin may effect the peritoneal interstitium-an important barrier of fluid and solutes transport.

Effects of chondroitin sulphate on fluid and solute transport during peritoneal dialysis in rats.

The effect of chondroitin sulphate (CS) on peritoneal fluid and solute transport was studied in rats undergoing peritoneal dialysis. In the presence of CS, net ultrafiltration increased, while absorption of glucose and horseradish peroxidase from the peritoneal cavity decreased. Albumin, used instead of CS, did not modify either fluid or solute transport. In in vitro experiments on isolated rabbit mesentery, CS decreased transmembrane water flow induced by hydrostatic pressure, and its effect was not fully reversed 60 minutes after "wash-out" of this glycosaminoglycan. We postulate that the polyanionic CS molecules are trapped in the peritoneal interstitium, thus decreasing its hydraulic conductivity and permeability, which in turn increases net fluid removal during peritoneal dialysis because of its slower absorption from the peritoneal cavity.

[Gentamicin may have an influence on the effectiveness of peritoneal dialysis]. / Gentamycyna moze miec wplyw na efektywnosc dializy otrzewnowej.

An effect of gentamicin on urea and uric acid transfer through peritoneum was studied in rabbits in vitro. It was found that gentamicin decreases urea transfer by approximately 30%. Its effect on uric acid transfer was transient so as mean values of this parameter did not differ statistically before and after antibiotic administration. It may be assumed that gentamicin decreases permeability of peritoneum for certain low molecules of endogenic origin. Such an effect may prove unfavourable when peritoneal dialysis efficiency is concerned.

[Effect of pyrazinoic acid on the transport function of the peritoneum--experimental studies]. / Wplyw kwasu pirazynowego na czynnosc transportowa otrzewnej--badania doswiadczalne.

Bidirectional transport of the urea, uric acid and albumin across parietal peritoneum was investigated in vitro under both normal conditions and following an administration of pyrazinoic acid. It was found that pyrazinoic acid decreased urea and uric acid transport from interstitial to mesothelial surface of the peritoneal membrane but did not affect the transport in reverse direction. Bidirectional transport of albumin remained unchanged. The obtained results are similar to those reported earlier and suggest that pyrazinamide actions are partially selective.

Vasopressin-induced changes in permeability of peritoneal mesothelium for urea "in vitro".

Vasopressin (ADH) acts in humans mainly upon renal collecting tubules. By changing their water permeability it plays a key role in regulation of renal water excretion. Acting upon vascular smooth muscle cells, it causes vasoconstriction and raised arterial blood pressure. This hormone was also proven to cause constriction of cultured mesangial cels, it causes vasoconstriction and raised arterial blood pressure. This urea (Seldin, Giebisch 1985), to release the natriuretic hormone as well as to stimulate hepatic glycogenolysis (Abramov et al. 1987). The influence of vasopressin upon peritoneal transport of solutes was studied, too. ADH influenced the passage of phosphate and rubidium through the isolated rabbit mesentery (Berndt, Gosselin 1961) as well as sodium flux through isolated rabbit omentum (Shear et al. 1966). It caused the drop in urea dialysance in dogs subjected to peritoneal dialysis (Henderson et al. 1971). The subject of our study was the assessment of the action of the antidiuretic hormone under "in vitro" conditions upon the peritoneal transfer of urea, the solute present in human body fluids and removable by peritoneal dialysis.

Effect of furosemide on transperitoneal passage of uric acid--experimental studies.

Application of furosemide (F) during peritoneal dialysis had been proposed for enlargement of peritoneal excretion of uric acid (UA), however, localization and mechanism of action of the drug remained obscure. To clarify the problem experimental studies on isolated peritoneum were performed with intention to establish whether and how transperitoneal flux of UA can be modified by F. Experiments were carried out in vitro on fragments of peritoneum taken from rabbits: parietal peritoneum and mesentery were isolated with single layer of mesothelium, and minimal amount of submesothelial tissue. Gradient of UA across the membranes was set up at 0.9-1.1 mmol/l, and flow estimated before and after addition of F (final conc. 10(-4) M) to the medium bathing either side of the membranes. Some differences in the flow between both types of the membranes as quantitative (greater flow in the parietal peritoneum), and qualitative (opposite succession of changes after F) were observed, however, the reaction for the diuretic was significant in both series of the experiments. The results confirm the suggestion that F can affect peritoneal excretion of UA during the dialysis, but at the same time they show that the mechanisms regulating the transperitoneal UA passage are complex.