Isolated heart preparations were prepared from guinea-pigs passively sensitized with mouse anti-DNP monoclonal IgE to study hemodynamic parameters and histamine release upon antigen challenge (DNP-BSA). In the best experimental conditions a triphasic response (increase, decrease, increase) in heart rate and in coronary flow occurred during 10 minutes after challenge. Coronary flow dramatically decreased (50%) at 2.5 min flanked by two periods of increase in flow and tachycardia (+ 55% of the basal heart rate). Contractile force decreased and remained at 80% of its initial value. Histamine is released within 2 minutes after antigen injection. After this period, the observed hemodynamic modifications seem to be due to other anaphylactic mediators such as prostaglandins, leukotrienes and most probably paf-acether.

The toxic role of numerous compounds accumulated in the body fluids of uremic patients is not well established. Instead of comparing the overall chromatographic plasma pattern of uremic patients to that of healthy subjects (1), one uremic symptom, namely active recent polyneuritis, was chosen and the correlation between the evolution of symptoms and biochemical patterns as modified by hemodialysis was evaluated. This correlation was complemented by a bioassay in which the fraction isolated previously exhibits a specific in vitro activity similar to that observed in vivo. These clinical, biochemical and bioassay techniques each contribute to form a correlative approach (2). Uremic polyneuritis was chosen to test the “middle molecule hypothesis” because it was found to respond favorably to any method which increases middle molecule transfer, i.e., more frequent or more prolonged dialysis or the use of a more porous membrane (3). In six patients with progressive and complete motor nerve paraplegia due to inadequate dialysis, the rapid improvement of nervous symptoms when using a dialysis membrane highly permeable to middle molecules brought us the first clinical evidence for the validity of the hypothesis (4).

Isolated perfused rat kidneys were passively sensitized by addition of either mouse ascitic fluid containing monoclonal IgE against dinitrophenol (DNP) or DNP-specific purified IgE. After washing the organ, defined doses of DNP-bovine serum albumin were given as bolus injection via the kidney artery. Antigen challenge of IgE-sensitized kidneys resulted in a dose-dependent increase of perfusion pressure starting with 5 micrograms antigen (2.46 +/- 0.2 mm Hg) and reaching a maximum at dose higher than 100 micrograms (10.3 +/- 1.6 mm Hg) (N = 4, means +/- 1 SD). A decrease of glomerular filtration rate was also observed which reached a plateau at 100 micrograms antigen (-68.5 +/- 2.9%) (N = 4). Regardless of the dose of antigen used, the urinary protein excretion markedly increased for the first five minutes following antigen injection and returned to basal values after 10 minutes. The total amounts of histamine, PGE2 and paf-acether (platelet-activating factor) released upon antigen challenge (1 mg) for 15 minutes reached maximal values of 405 +/- 21.1 ng, 286 +/- 19.4 pg and 12.3 +/- 3.2 ng (N = 5), respectively. None of these hemodynamic and biochemical effects were observed using IgG1 monoclonal antibodies or when the ascitic fluid containing monoclonal IgE used to sensitize the organ was heated at 56 degrees C for two hours. Thus, we have described a pure IgE-dependent rat kidney anaphylaxis. Antigen challenge markedly altered renal parameters and triggered the release of various mediators from the organ, suggesting that type I-hypersensitivity reactions may play a role in renal pathophysiology.

Fractions containing substances weighing less than 1800 daltons have been obtained from hemofiltrate and peritoneal dialysis fluid. Oligosaccharides and glycopeptides were evidenced in these fractions by paper chromatography. This analytical procedure has been applied to a fraction containing peak b4-2 obtained by molecular exclusion chromatography on Sephadex G-15 followed by ion exchange chromatography on Sephadex A-25 (Cueille et al.). Preparative paper chromatography of this fraction evidenced 3 carbohydrate and 3 peptidic fractions. Study of the carbohydrate fraction (01, 03, 04) by gas-chromatography and mass spectrometry shows that they are composed of glucuronoconjugates whose aglycons have not been identified. Fraction 04 which contains subpeak b4-2 has been found to have an inhibitory effect on the action potentials of the sural nerve of the frog.

ABSTRACT Among the middle molecule fractions obtained by high performance gel chromatography on Sephadex G‐15 combined with gradient ion exchange chromatography on DEAE Sephadex A‐25 from plasma ultrafiltrates of six polyneuropathic patients, only peak b 4–2 was at a significantly higher concentration than that obtained from uremic patients with neuropathy. Purification of the b 4–2 solute allows its quantitative determination in biological fluids. The b 4–2 plasma concentrations are 1 mg/L in healthy subjects (n=30), 4.6 ± 0.2 mg/L in uremic patients (n=67) and 13–19 mg/L in six polyneuropathic patients. The 24‐hr urinary excretion is 13 ± 1 mg. The weekly removal rates in hemofiltration or in hemodialysis, using high permeability membrane three times a week, are 38–40 mg. In an in vitro sural nerve test for the evaluation of middle molecule neurotoxicity, the b 4–2 solute exhibits a neurotoxic effect at concentrations similar to those found in plasma of neuropathic patients. Preliminary results of an attempt to identify the neurotoxin indicate that it is an acid‐polyo derivative.

In order to evaluate the in vitro neurotoxicity of MM fractions obtained by chromatographic analytical methods from plasma of uremic polyneuropathic patients and urine of healthy subjects, we performed an in vitro test on isolated frog sural nerve. The nerve is incubated in various media and stimulated by a rectangular shock (0.05ms, 1Hz, intensity supramaximal). Action potential are recorded. Results were expressed by the inhibition index Ii = 1 divided by t 1/2 x 100 where t 1/2 is the time (in min) necessary to reduce the 1/2 the spike amplitude. When the nerve is immerged in Ringer solution or plasma of healthy subjects Ii congruent to 0. Among MM fractions obtained by gel chromatography Sephadex G-15 from uremic polyneuropathic subjects' plasma and healthy subjects' urine, only fraction b gives a positive response (Ii congruent to 0.86 +/- 0.02, n = 4). From the 6 sub-fractions obtained by anion exchange chromatography DEAE Sephadex A-25 of fraction b, only sub-fraction b4-2 exhibits a reduction of the spike amplitude, Ii = 0.54 when concentration b4-2 is 6 mg/L, Ii = 1.30 when b4-2 plasma concentration is 14 mg/L. These values increase with the severity of neurologic symptoms. A positive correlation was found between Ii and various b4-2 concentrations of standard solutions and plasma ultrafiltrate (y = 0.089x + 0.046, r = 0.890, n = 13). In vitro sural nerve test demonstrated its sensitivity as a tool for monitoring analytical and preparative procedures developed for isolation of neurotoxic solutes.

Impaired weight gain, a common feature in experimental chronic uremia, led to compare oxygen consumtion values of chronic uremic rats and their controls. Oxygen consumption measurement may allow an approach to the evaluation of total energy requirement. Three groups of animals were considered: uremic rats (CU) of less weight (402.66 +/- 9.9 g) than their control group (NA) (498.3 +/- 26.13) and younger normal animals (NJ), showing a similar weight (376.0 +/- 8.8) to that of the uremics. Significant differences in oxygen consumption values/body weight were found between group NJ (0.927 +/- 0.042 ml O2/mn/100 g rat) and both groups CU and NA (0.788 +/- 0.036 and 0.788 +/- 0.028 respectively). Proportional decrease of energetically active body mass in chronic uremic animals may be the reason of the findings.

Purification of b4-2 sub-peak obtained on DEAE Sephadex A25 chromatography gave us the possibility of quantifying the plasma concentration of the neurotoxin present in uraemic patients with active polyneuropathy. From the purified neurotoxin isolated by kieselguhr and cellulose chromatography we calibrated analytic columns for b4-2 analysis. Plasma concentration, measured in 6 uraemic neuropathic patients, is between 13 and 19 mg/litre. In 52 uraemic patients without neuropathy, the plasma concentration is between 3 and 9 mg/litre. In 20 healthy subjects the plasma concentration is less than 1 mg/litre. The weekly neurotoxin removal in uraemic patients without neuropathy, treated by a five hours RP6 session 3 times a week, is of the same order of magnitude as the weekly urinary excretion in healthy subjects. Preliminary results of a tentative identification of this purified product indicate that it is not a polypeptide but an acid-polyol with carbohydrate structure.

Experimental chronic renal failure in the rat with reduced and stable glomerular filtration rate has been previously studied, and several models have been described. Most of the studies have used techniques which attempt to reduce renal function by means of reducing blood flow [1–3], by inducing perinephritis [4], by surgically reducing the parenchyma [5–10], or by papillectomy [7, 11]. Diminution by surgery seems to be the technique most often chosen because of the possibilities of quantification which it affords. The use of this technique does, however, entail the risk of hemorrhage. This paper describes our attempt to devise a simple technique for inducing an advanced, stable chronic renal failure without such postoperative complications.