Bengt Rippe

Transendothelial transport: the vesicle controversy.

The relative contribution of transcytosis vs. large pore transport to the passage of macromolecules across microvascular endothelia has been a controversial issue for nearly half a century. To separate transcytosis from ‘porous’ transport, the transcytosis inhibitors N-ethylmaleimide (NEM) and filipin have been tested in in situ or ex vivo perfused organs with highly conflicting results. In continually weighed isolated perfused organs, where measurements of pre- and post-capillary resistances, capillary pressure and capillary filtration coefficients can be repeatedly performed, high doses of NEM and filipin increased the bulk transport of macromolecules from blood to tissue, despite producing vasoconstriction. By contrast, in in situ perfused organs, marked reductions in the tissue uptake of albumin tracer have been observed after NEM and filipin. When tissue cooling has been employed as a means of inhibiting (active) transcytosis, results have invariably shown a low cooling sensitivity of albumin transport, compatible with passive transendothelial passage of albumin. This observation is further strengthened by the commonly observed dependence of albumin transport upon the capillary pressure and the rate of transcapillary convection. For low-density lipoprotein (LDL), a cooling-sensitive, non-selective transport component has been discovered, which may be represented by filtration through paracellular gaps, lateral diffusion through transendothelial channels formed by fused vesicles, or by transcytosis. From a physiological standpoint there is little evidence supporting active transendothelial transport of most plasma macromolecules. This seems to be supported by studies on caveolin-1-deficient mice lacking plasmalemmal vesicles (caveolae), in which there are no obvious abnormalities in the transendothelial transport of albumin, immunoglobulins or lipoproteins. Nevertheless, specific transport in peripheral capillaries of several hormones and other specific substances, similar to that existing across the blood-brain barrier, still remains as a possibility.

Peritoneal physiology-transport of solutes

The peritoneal cavity, which lodges the abdominal viscera, represents the largest serosal cavity in the body. Functionally it may be regarded as a large continuous interstitial space. It is lined by a thin, translucent membrane, the peritoneum, which covers the inner surface of the abdominal wall (parietal peritoneum) and the majority of visceral organs (visceral peritoneum) and which forms omenta. The lining structure of the peritoneal cavity is usually denoted the ‘peritoneal membrane’. Because of its relatively large surface area (1-2 m2 in adults) [1], its high dregree of capillarization and relatively high blood flow, it can be rather effectively utilized as an endogenous dialysis membrane to remove uremic toxins and water from the body fluids of patients with end-stage renal disease during peritoneal dialysis (PD). The peritoneal cavity is also suitable as a portal entry for various drugs and hormones administered intraperitoneally (i.p.). In peritonitis (during PD), for example, antibiotics can be given i.p. for systemic use, because of the high transport capacity of the peritoneal membrane.

Intraperitoneal fluid volume changes during peritoneal dialysis in the rat: indicator dilution vs. volumetric measurements.

In order to validate the single injection RISA (125I human serum albumin) indicator diluation technique for assessing the alterations in intraperitoneal (i.p.) dialysate volume (IPV) which occur vs. time [V(t)] during peritoneal dialysis (PD), the RISA dilution technique was compared to V(t) determinations using a direct volume recovery method in Wistar rats. Sixteen milliliters of either 1.36 or 3.86% Dianeal or 0.9% NaCl were used as dialysis fluids in exchanges lasting between 1 and 360 min. Approximately 4% (4.41 +/- 0.59 (SE; n = 8) for 1.36% Dianeal and 4.07 +/- 0.72 (n = 4) for 3.86% Dianeal) of the RISA dose given intraperitoneally was lost from the dialysate during the first 1(-1.5) min after instillation, conceivably due to rapid tracer adsorption to peritoneal surfaces. Following the initial instant tracer loss and RISA dilution due to a residual volume (3.07 +/- 0.18 ml; n = 12), RISA disappeared at a fractional rate (FDR) of 2.10 +/- 0.14 x 10(-3) min-1 and 1.67 +/- 0.09 x 10(-3) min-1, during the first 30 min for 1.36 and 3.86% Dianeal, respectively. The overall FDR was 1.33 +/- 0.10 x 10(-3) and 0.707 +/- 0.082 x 10(-3) min-1 for 1.36% Dianeal (0-150 min) and 3.86% Dianeal (0-360 min), respectively, while the overall (0-150 min) FDR for the NaCl exchanges was 1.40 +/- 0.21 x 10(-3) min-1. These values correspond to RISA clearances out of the peritoneal cavity (KE) of 29.2 +/- 1.8, 22.1 +/- 1.6, and 25.7 +/- 2.4 microliter x min-1 for 1.36 and 3.86% Dianeal and 0.9% NaCl, respectively. The KE value for 3.86% Dianeal was significantly (p < 0.05) lower than for the two dialysates with lower osmolality. The slightly enhanced FDR of RISA during the first 30 min was partly due to the presence of nonprotein-bound free iodine in the RISA preparation used, and also to an enhanced disappearance of albumin during the first portion of the dwell. V(t) data from individual experiments using the RISA dilution technique (RISA curves) were analyzed by computer-aided nonlinear least-squares regression analysis.(ABSTRACT TRUNCATED AT 400 WORDS)

The effects of low-sodium peritoneal dialysis fluids on blood pressure, thirst and volume status

Background. Poor ultrafiltration is associated with worse outcomes in peritoneal dialysis (PD) patients. This might in part reflect problems associated with salt and water excess. Increasing the diffusive component of peritoneal sodium removal using low-sodium PD fluids might have beneficial effects on blood pressure (BP), thirst and fluid status that could translate into clinical benefits. Methods. Using a multicentre, prospective, baseline controlled (1 month), non-randomized intervention (2 months) design, two novel solutions designed from predictions using the three-pore model were investigated. In group A ([Na+] = 115 mmol/l), the glucose (G) was increased to 2.0% to compensate for reduced osmolality whereas in group B ([Na+] = 102 mmol/l), it was unchanged (2.5%). Both solutions were substituted for one 3- to 5-h exchange per day and no change was made to the rest of the dialysis regime. Results. Ten patients in group A and 15 in group B completed the study. Both solutions resulted in significant increases (30–50 mmol/dwell) in diffusive sodium removal during the test exchanges, P < 0.001. Ultrafiltration was maintained in group A but reduced in group B. Ambulatory nocturnal mean BP fell in group A [93.1 ± 10.6 mmHg (±SD) versus 85.1 ± 10.2 mmHg, P < 0.05], but was stable in group B (95.4 ± 9.4 versus 95.1.1 ± 10.7 mmHg, NS). Thirst reduced independent of appetite and mood in both groups by 2 months, more markedly in group A. Indices of fluid status, including TBW by bioimpedance and D dilution also improved in group A, P < 0.05, whereas weight increased in group B. Conclusions. Increasing the diffusive component of sodium removal whilst maintaining ultrafiltration is associated with improvements in BP, thirst and fluid status. The lack of effect seen with uncompensated low-sodium dialysate suggests that these benefits cannot be achieved by manipulation of dialysate sodium removal alone. These observations provide valuable information of the design of future randomized studies to establish the clinical role for low-sodium dialysis fluids.

Glomerular sieving of three neutral polysaccharides, polyethylene oxide and bikunin in rat. Effects of molecular size and conformation.

Aim:  Polysaccharides and many other non‐protein polymers generally have a more open, flexible and asymmetrical structure compared with globular proteins. For a given molecular weight (MW), the Stokes–Einstein radius (ae) of the following polymers increases in the order: Ficoll < dextran ≤ pullulan < polyethylene oxide (PEO). We have tested the hypothesis that such an increase in ‘molecular extension’ will increase the molecule’s glomerular permeability. Thus, we investigated the glomerular sieving coefficients (θ) of the mentioned polymers and of the negatively charged and extended protein bikunin.

The Peritoneal Microcirculation in Peritoneal Dialysis

This paper deals with the peritoneal microcirculation and with peritoneal exchange occurring in peritoneal dialysis (PD). The capillary wall is a major barrier to solute and water exchange across the peritoneal membrane. There is a bimodal size‐selectivity of solute transport between blood and the peritoneal cavity, through pores of radius ∼40–50 Å as well as through a very low number of large pores of radius ∼250 Å. Furthermore, during glucose‐induced osmosis during PD, nearly 40% of the total osmotic water flow occurs through molecular water channels, termed “aquaporin‐1.” This causes an inequality between 1−σ and the sieving coefficient for small solutes, which is a key feature of the “threepore model” of peritoneal transport. The peritoneal interstitium, coupled in series with the capillary walls, markedly modifies small‐solute transport and makes large‐solute transport asymmetric. Thus, although severely restricted in the blood‐to‐peritoneal direction, the absorption of large solutes from the peritoneal cavity occurs at a high clearance rate (∼1 mL/min), largely independent of molecular radius. True absorption of macromolecules to the blood via lymphatics, however, seems to be occurring at a rate of ∼0.2 mL/min. Several controversial issues regarding transcapillary and transperitoneal exchange mechanisms are discussed in this paper.

Acute hyperglycemia induces rapid, reversible increases in glomerular permeability in nondiabetic rats

This study was performed to investigate the impact of acute hyperglycemia (HG) on the permeability of the normal glomerular filtration barrier in vivo. In anesthetized Wistar rats (250-280 g), the left ureter was catheterized for urine collection, while simultaneously blood access was achieved. Rats received an intravenous (iv) infusion of either 1) hypertonic glucose to maintain blood glucose at 20-25 mM (G; n = 8); 2) hypertonic glucose as in 1) and a RhoA-kinase inhibitor (Y-27632; Rho-G; n = 8); 3) 20% mannitol (MANN; n = 7) or 4) hypertonic (12%) NaCl to maintain plasma crystalloid osmotic pressure (pi(cry)) at approximately 320-325 mosmol/l (NaCl; n = 8) or 5) physiological saline (SHAM; n = 8). FITC-Ficoll 70/400 was infused iv for at least 20 min before termination of the experiments, and plasma and urine were collected to determine the glomerular sieving coefficients (theta) for polydisperse Ficoll (molecular radius 15-80 A) by high-performance size-exclusion chromatography. In G there was a marked increase in for Ficoll(55-80A) at 20 min, which was completely reversible within 60 min and abrogated by a Rho-kinase (ROCK) inhibitor, while glomerular permeability remained unchanged in MANN and NaCl. In conclusion, acute HG caused rapid, reversible increases in for large Ficolls, not related to the concomitant hyperosmolarity, but sensitive to ROCK inhibition. The changes observed were consistent with the formation of an increased number of large pores in the glomerular filter. The sensitivity of the permeability changes to ROCK inhibition strongly indicates that the cytoskeleton of the cells in the glomerular barrier may be involved in these alterations.

A1M/α1-Microglobulin Protects from Heme-Induced Placental and Renal Damage in a Pregnant Sheep Model of Preeclampsia.

Preeclampsia (PE) is a serious pregnancy complication that manifests as hypertension and proteinuria after the 20th gestation week. Previously, fetal hemoglobin (HbF) has been identified as a plausible causative factor. Cell-free Hb and its degradation products are known to cause oxidative stress and tissue damage, typical of the PE placenta. A1M (α1-microglobulin) is an endogenous scavenger of radicals and heme. Here, the usefulness of A1M as a treatment for PE is investigated in the pregnant ewe PE model, in which starvation induces PE symptoms via hemolysis. Eleven ewes, in late pregnancy, were starved for 36 hours and then treated with A1M (n = 5) or placebo (n = 6) injections. After injections, the ewes were re-fed and observed for additional 72 hours. They were monitored for blood pressure, proteinuria, blood cell distribution and clinical and inflammation markers in plasma. Before termination, the utero-placental circulation was analyzed with Doppler velocimetry and the kidney glomerular function was analyzed by Ficoll sieving. At termination, blood, kidney and placenta samples were collected and analyzed for changes in gene expression and tissue structure. The starvation resulted in increased amounts of the hemolysis marker bilirubin in the blood, structural damages to the placenta and kidneys and an increased glomerular sieving coefficient indicating a defect filtration barrier. Treatment with A1M ameliorated these changes without signs of side-effects. In conclusion, A1M displayed positive therapeutic effects in the ewe starvation PE model, and was well tolerated. Therefore, we suggest A1M as a plausible treatment for PE in humans.

Water transport across the peritoneal membrane

Peritoneal dialysis involves diffusive and convective transports and osmosis through the highly vascularized peritoneal membrane. The capillary endothelium offers the rate-limiting hindrance for solute and water transport. It can be functionally described in terms of a three-pore model including transcellular, ultrasmall pores responsible for free-water transport during crystalloid osmosis. Several lines of evidence have demonstrated that the water channel aquaporin-1 (AQP1) corresponds to the ultrasmall pore located in endothelial cells. Studies in Aqp1 mice have shown that deletion of AQP1 is reflected by a 50% decrease in ultrafiltration and a disappearance of the sodium sieving. Haploinsufficiency in AQP1 is also reflected by a significant attenuation of water transport. Conversely, studies in a rat model and in PD patients have shown that the induction of AQP1 in peritoneal capillaries by corticosteroids is reflected by increased water transport and ultrafiltration, without affecting the osmotic gradient and small-solute transport. Recent data have demonstrated that a novel agonist of AQP1, predicted to stabilize the open-state conformation of the channel, modulates water transport and improves ultrafiltration. Whether increasing the expression of AQP1 or gating the already existing channels would be clinically useful in PD patients remains to be investigated.

Acute and sustained actions of hyperglycaemia on endothelial and glomerular barrier permeability

Microalbuminuria is an established marker of systemic endothelial dysfunction, which for patients with diabetes signals an increased risk of both diabetic nephropathy and cardiovascular complications. A better understanding of the pathogenesis of microalbuminuria is important in the quest of finding new approaches to treat patients with diabetes. Direct acute effects of episodes of hyperglycaemia (HG) could have implications for the microalbuminuria seen in early diabetes before renal structural alterations have started, especially in those patients with poor glycaemic control. This review summarizes the literature evidence that acute or sustained HG may lead to an increased vascular or glomerular permeability. Special focus is on glomerular barrier permeability. There is evidence in the literature that HG increases systemic capillary and glomerular barrier permeability within 20–30 min in vivo in rats and mice. Furthermore, exposure of monolayers of cultured endothelial cells to HG has been shown to increase monolayer permeability rapidly and transiently (during 60–100 min). Instant cellular changes following F‐actin cytoskeleton rearrangements, which could be abrogated by Rho‐kinase (ROCK) inhibition, are implicated. Data in this review also suggest that activation of protein kinase C, the polyol pathway, and an increased release of reactive oxygen species (ROS) and cytokines could contribute to the increase in barrier permeability induced by HG. Recent in vitro data from cultured podocyte monolayers also designates a role of insulin in acute podocyte F‐actin remodelling, underpinning the complexity of the mechanisms leading to glomerular and endothelial barrier alterations in diabetes mellitus.