Effect of a silver device in preventing catheter-related infections in peritoneal dialysis patients: silver ring prophylaxis at the catheter exit study.

Catheter-related infections remain a significant cause of method failure in chronic peritoneal dialysis (PD) therapy. Given the increasing antibiotic resistance, such nonpharmacological strategies as local silver devices attract more interest. To establish whether a silver ring device (designed by Grosse-Siestrup in 1992) mounted onto the PD catheter and placed at the exit site at skin level is effective in preventing exit-site and other catheter-related infections, a prospective 12-month, multicenter, controlled study stratified by diabetes status was conducted. The study subjects were assessed by an extensive structured inventory, including a broad spectrum of control variables, such as age, body mass index (BMI), Staphylococcus aureus carrier status, catheter features, mode and quality of PD therapy, comorbidity, and psychosocial rehabilitation. Ten experienced German outpatient dialysis centers (seven adult, three pediatric) participated in the trial. All eligible patients (n=195) from the study area without catheter-related infections during the ascertainment period were included (incidental subjects undergoing PD therapy for at least 3 months). The main outcome measures were the occurrence of first exit-site infections (primary study end point), sinus tract/tunnel infection, and peritonitis. Ninety-seven patients were assigned to the silver ring and 98 patients to the control group. Baseline characteristics of age, sex, proportion of pediatric and incidental patients, S aureus carrier status, and other variables were similar in both groups. The incidence of infections in the silver ring group versus the control group was as follows: 23 of 97 versus 16 of 98 patients had exit-site infections, 12 of 97 versus 12 of 98 patients had sinus tract/tunnel infections, 16 of 97 versus 18 of 98 patients had peritonitis, respectively. Kaplan-Meier analysis for the probability of an infection-free interval showed no statistical difference (log-rank test) between the two groups. Displacement of the silver ring contributed to study termination in 6% of the study group patients, including two patients with catheter loss. Univariate analysis and multiple logistic regression identified younger age (<50 years), low serum albumin level (<35 g/L), number of previously placed PD catheters, short cuff-exit distance (<2 cm), and S aureus nasal carriage as risk factors for the development of exit-site infections. In conclusion, our study does not show any benefit of the silver ring in preventing catheter-related infections in PD patients. Thus, prevention of infection-related method failure in PD still has to rely on conventional antibiotic treatment strategies and less so on alternative methods.

Cell transformation induces a cytoplasmic Ca2+ oscillator in Madin-Darby canine kidney cells.

Alkaline stress transforms Madin-Darby canine kidney (MDCK) cells as indicated by loss of epithelial structure, multilayer cell growth and formation of foci. In the present study we report that transformed MDCK cells (MDCK-F cells) exhibit spontaneous and lasting oscillations of intracellular Ca2+ concentration ([Ca2+]i), which are absent in non-transformed cells. Oscillations, as revealed by Fura-2 video imaging, were due to the activity of an inositol 1,4,5-trisphosphate-(InsP3)-sensitive Ca2+ store since their frequency was dependent on bradykinin concentration and they were abolished by the phosphoinositidase C inhibitor U73122. Moreover, blockers of the cytoplasmic Ca(2+)-ATPase, thapsigargin and 2,5-di-(tetr-butyl)-1,4-benzohydroquinone inhibited oscillatory activity. In contrast, neither injection of ruthenium red, ryanodine nor caffeine had any effect on oscillations. Analysis of the spatial distribution of [Ca2+]i showed that Ca2+ transients originated from an initiation site constant for a given cell and spread through the cell as an advancing Ca2+ wave. Oscillations started in a random manner from single cells and spread over neighbouring cells, suggesting a kind of intercellular communication. We conclude that MDCK-F cells have acquired the ability for endogenous Ca2+ release through transformation. Oscillations are primarily due to the activity of an InsP3-sensitive cytosolic Ca2+ oscillator.

Spontaneously oscillating K+ channel activity in transformed Madin-Darby canine kidney cells.

Intracellular alkalinization is known to be associated with tumorigenic transformation. Besides phenotypical alterations alkali-transformed Madin-Darby canine kidney (MDCK) cells exhibit a spontaneously oscillating cell membrane potential (PD). Using single-channel patch clamp techniques, it was the aim of this study to identify the ion channel underlying the rhythmic hyperpolarizations of the PD. In the cell-attached patch configuration, we found that channel activity was oscillating. The frequency of channel oscillations is 1.1 +/- 0.1 min-1. At the peak of oscillatory channel activity, single-channel current was -2.7 +/- 0.05 pA, and in the resting state it was -1.95 +/- 0.05 pA. Given the single-channel conductance of 53 +/- 3 pS for inward (and of 27 +/- 5 pS for outward) current the difference of single-channel current amplitude corresponded to a hyperpolarization of approximately 14 mV. The channel is selective for K+ over Na+. Channel kinetics are characterized by one open and by three closed time constants. The channel is Ca2+ sensitive. Half maximal activation in the inside-out patch mode is achieved at a Ca2+ concentration of 10 mumol/liter. In addition, we also found a 13-pS K+ channel that shows no oscillatory activity in the cell-attached patch configuration and that was not Ca2+ sensitive. We conclude that the Ca(2+)-sensitive 53-pS K+ channel is underlying spontaneous oscillations of the PD. It has virtually identical biophysical properties as a Ca(2+)-sensitive K+ channel in nontransformed parent MDCK cells. Hence, alkali-induced transformation of MDCK cells did not affect the channel protein itself but its regulators thereby causing spontaneous fluctuations of the PD.

Oscillations: a key event in transformed renal epithelial cells.

Intracellular pH (pHi) plays a critical role in the entry of cells into the DNA-synthesis phase of the cell cycle. Alterations in pHi may contribute to abnormal proliferative responses such as those seen in tumorigenic cells. We observed that alkaline stress leads to genomic transformation of Madin-Darby canine kidney (MDCK) cells. Transformed cells (F cells) form "foci" in culture, lack contact inhibition, and are able to migrate, typical characteristics of dedifferentiated tumorigenic cells. F cells exhibit spontaneous biorhythmicity. Rhythmic transmembrane Ca2+ flux activates plasma membrane K+ channels and Na+/H+ exchange. This leads to periodic changes of membrane voltage and pHi at about one cycle per minute. We conclude that endogenous oscillatory activity could be a trigger mechanism for DNA synthesis, proliferation, and abnormal growth of renal epithelial cells in culture.

Spontaneous membrane potential oscillations in Madin-Darby canine kidney cells transformed by alkaline stress.

High pH is known to be associated with normal cell growth and neoplastic transformation. We observed that Madin-Darby canine kidney (MDCK) cells grown under sustained alkaline stress (pH 7.7) develop "foci" composed of spindle-shaped cells lacking contact inhibition and exhibiting only poor adhesion to the culture support. Foci-developing (F) cells were cloned and grown in control medium (pH 7.4), where they maintained their neoplastic features indicating a stable pH-induced genetic transformation. After F cells had been fused to giant cells with polyethylene glycol, the cell membrane potential (Vm) was measured by means of microelectrodes. In contrast to non-transformed MDCK cells, Vm of F cells showed spontaneous biorhythmicity caused by periodic opening of Ca2(+)-activated K+ channels. Spiking activity was blunted by the Ca2+ channel blocker nifedipine, by the K+ channel blocker Ba2+, by the Na+/H+ exchange blocker amiloride and its analogue ethylisopropylamiloride, and by an extracellular pH of 7.6 and 6.8. We conclude that MDCK cells transformed by sustained alkaline stress have lost their stable plasma membrane potential but, instead, exhibit endogenous Ca2(+)- and pH-sensitive oscillations.

Alkaline stress transforms Madin-Darby canine kidney cells.

Similar to growth factors aldosterone stimulates Na+/H+ exchange in renal target cells leading to cytoplasmic alkalinization. An alkaline intracellular pH reduces the H+ bonds between repressor proteins and DNA leading to the destabilization of the nuclear chromatin. We observed that sustained alkaline stress "per se" can lead to malignant transformation of Madin-Darby canine kidney (MDCK) cells. Cells grown for two weeks in alkaline culture medium (pH 7.8) developed multiple "foci" composed of spindle-shaped pleomorphic cells lacking contact inhibition and exhibiting poor adhesion to the culture support, typical characteristics of dedifferentiated tumor cells. "Focus" cells were cloned and grown in standard medium (pH 7.4). Cells maintained their abnormal growth pattern, indicating stable pH-induced genetic transformation. Cells were fused with polyethylene glycol to giant cells and impaled with microelectrodes. In contrast to non-transformed giant MDCK cells the plasma membrane potential showed spontaneous oscillations that could be virtually abolished by the omission of extracellular Ca2+ or by the addition of the K+ channel blocker Ba2+. We conclude that sustained alkaline stress can induce malignant transformation in MDCK cells indicated by an abnormal growth pattern and by membrane potential oscillations most likely due to Ca2+ activated K+ channels in the plasma membrane.

Electrode sandwich technique: a method for studying K+ transport in renal cells.

Distal tubules were harvested from frog kidney and placed on the membrane of a K+-selective macroelectrode. Then the renal tissue was covered with a dialysis membrane to produce a closed extracellular compartment with a constant volume (40 microliter). K+ fluxes in and out of the cells could be determined, since the steady-state K+ activity during constant perfusion changed to a new steady state when perfusion was stopped. Inhibition of passive K+ permeability by the addition of Ba2+ resulted in K+ uptake by the cells because of the function of the Na+-K+ pump. Inhibition of the pump by the addition of ouabain led to K+ efflux from cells reflecting the passive K+ permeability. Because K+ net movement under control conditions (no Ba2+ or ouabain) results from both uptake and efflux, subtraction of K+ uptake (in the presence of Ba2+) from control K+ net flux reveals the passive K+ efflux. This value agrees well with that obtained with ouabain. Furosemide led to a significant K+ shift from the extracellular compartment into the intracellular compartment. Reduction of extracellular pH from 7.8 to 6.0 decreased the rate of K+ uptake by 39 +/- 7% and the K+ leak by 51 +/- 11%. We conclude that K+ uptake and K+ release can be functionally separated. This so-called "electrode sandwich technique" permits evaluation of pump and leak independently in the same cell population.

Aldosterone activates Na+/H+ exchange and raises cytoplasmic pH in target cells of the amphibian kidney.

The hypothesis was tested if the mineralocorticoid hormone aldosterone stimulates Na+/H+ exchange in "giant cells" fused from individual target cells of the distal nephron of the frog kidney. By means of microelectrodes, steady-state intracellular pH (pHi) and pHi recovery from an acid load were recorded continuously while the fused cells were exposed to aldosterone. Twenty minutes after addition of the hormone, pHi started to rise and reached a new steady state after about 60 min (delta pHi = 0.28 +/- 0.01). After hormone treatment, pHi recovered significantly faster in response to an intracellular acid load. The diuretic drug amiloride blocked pHi recovery. Experiments in intact tubules showed that aldosterone induces H+ and K+ secretion. Thus, intracellular alkalosis, mediated by Na+/H+ exchange, could serve as a signal that activates pH-sensitive K+ channels of the luminal cell membrane.