Regulation of macrophage motility by the water channel aquaporin-1: crucial role of M0/M2 phenotype switch.

The water channel aquaporin-1 (AQP1) promotes migration of many cell types. Although AQP1 is expressed in macrophages, its potential role in macrophage motility, particularly in relation with phenotype polarization, remains unknown. We here addressed these issues in peritoneal macrophages isolated from AQP1-deficient mice, either undifferentiated (M0) or stimulated with LPS to orientate towards pro-inflammatory phenotype (classical macrophage activation; M1). In non-stimulated macrophages, ablation of AQP1 (like inhibition by HgCl2) increased by 2-3 fold spontaneous migration in a Src/PI3K/Rac-dependent manner. This correlated with cell elongation and formation of lamellipodia/ruffles, resulting in membrane lipid and F4/80 recruitment to the leading edge. This indicated that AQP1 normally suppresses migration of resting macrophages, as opposed to other cell types. Resting Aqp1-/- macrophages exhibited CD206 redistribution into ruffles and increased arginase activity like IL4/IL13 (alternative macrophage activation; M2), indicating a M0-M2 shift. In contrast, upon M1 orientation by LPS in vitro or peritoneal inflammation in vivo, migration of Aqp1-/- macrophages was reduced. Taken together, these data indicate that AQP1 oppositely regulates macrophage migration, depending on stimulation or not by LPS, and that macrophage phenotypic and migratory changes may be regulated independently of external cues.

Cystic gene dosage influences kidney lesions after nephron reduction.

Cystic kidney disease is characterized by the progressive development of multiple fluid-filled cysts. Cysts can be acquired, or they may appear during development or in postnatal life due to specific gene defects and lead to renal failure. The most frequent form of this disease is the inherited polycystic kidney disease (PKD). Experimental models of PKD showed that an increase of cellular proliferation and apoptosis as well as defects in apico-basal and planar cell polarity or cilia play a critical role in cyst development. However, little is known about the mechanisms and the mediators involved in acquired cystic kidney diseases (ACKD). In this study, we used the nephron reduction as a model to study the mechanisms underlying cyst development in ACKD. We found that tubular dilations after nephron reduction recapitulated most of the morphological features of ACKD. The development of tubular dilations was associated with a dramatic increase of cell proliferation. In contrast, the apico-basal polarity and cilia did not seem to be affected. Interestingly, polycystin 1 and fibrocystin were markedly increased and polycystin 2 was decreased in cells lining the dilated tubules, whereas the expression of several other cystic genes did not change. More importantly, Pkd1 haploinsufficiency accelerated the development of tubular dilations after nephron reduction, a phenotype that was associated to a further increase of cell proliferation. These data were relevant to humans ACKD, as cystic genes expression and the rate of cell proliferation were also increased. In conclusion, our study suggests that the nephron reduction can be considered a suitable model to study ACKD and that dosage of genes involved in PKD is also important in ACKD.

AqF026 is a pharmacologic agonist of the water channel aquaporin-1.

Aquaporin-1 (AQP1) facilitates the osmotic transport of water across the capillary endothelium, among other cell types, and thereby has a substantial role in ultrafiltration during peritoneal dialysis. At present, pharmacologic agents that enhance AQP1-mediated water transport, which would be expected to increase the efficiency of peritoneal dialysis, are not available. Here, we describe AqF026, an aquaporin agonist that is a chemical derivative of the arylsulfonamide compound furosemide. In the Xenopus laevis oocyte system, extracellular AqF026 potentiated the channel activity of human AQP1 by >20% but had no effect on channel activity of AQP4. We found that the intracellular binding site for AQP1 involves loop D, a region associated with channel gating. In a mouse model of peritoneal dialysis, AqF026 enhanced the osmotic transport of water across the peritoneal membrane but did not affect the osmotic gradient, the transport of small solutes, or the localization and expression of AQP1 on the plasma membrane. Furthermore, AqF026 did not potentiate water transport in Aqp1-null mice, suggesting that indirect mechanisms involving other channels or transporters were unlikely. Last, in a mouse gastric antrum preparation, AqF026 did not affect the Na-K-Cl cotransporter NKCC1. In summary, AqF026 directly and specifically potentiates AQP1-mediated water transport, suggesting that it deserves additional investigation for applications such as peritoneal dialysis or clinical situations associated with defective water handling.

L-carnitine is an osmotic agent suitable for peritoneal dialysis.

Excessive intraperitoneal absorption of glucose during peritoneal dialysis has both local cytotoxic and systemic metabolic effects. Here we evaluate peritoneal dialysis solutions containing L-carnitine, an osmotically active compound that induces fluid flow across the peritoneum. In rats, L-carnitine in the peritoneal cavity had a dose-dependent osmotic effect similar to glucose. Analogous ultrafiltration and small solute transport characteristics were found for dialysates containing 3.86% glucose, equimolar L-carnitine, or combinations of both osmotic agents in mice. About half of the ultrafiltration generated by L-carnitine reflected facilitated water transport by aquaporin-1 (AQP1) water channels of endothelial cells. Nocturnal exchanges with 1.5% glucose and 0.25% L-carnitine in four patients receiving continuous ambulatory peritoneal dialysis were well tolerated and associated with higher net ultrafiltration than that achieved with 2.5% glucose solutions, despite the lower osmolarity of the carnitine-containing solution. Addition of L-carnitine to endothelial cells in culture increased the expression of AQP1, significantly improved viability, and prevented glucose-induced apoptosis. In a standard toxicity test, the addition of L-carnitine to peritoneal dialysis solution improved the viability of L929 fibroblasts. Thus, our studies support the use of L-carnitine as an alternative osmotic agent in peritoneal dialysis.

Expression patterns of the aquaporin gene family during renal development: influence of genetic variability.

High-throughput analyses have shown that aquaporins (AQPs) belong to a cluster of genes that are differentially expressed during kidney organogenesis. However, the spatiotemporal expression patterns of the AQP gene family during tubular maturation and the potential influence of genetic variation on these patterns and on water handling remain unknown. We investigated the expression patterns of all AQP isoforms in fetal (E13.5 to E18.5), postnatal (P1 to P28), and adult (9 weeks) kidneys of inbred (C57BL/6J) and outbred (CD-1) mice. Using quantitative polymerase chain reaction (PCR), we evidenced two mRNA patterns during tubular maturation in C57 mice. The AQPs 1-7-11 showed an early (from E14.5) and progressive increase to adult levels, similar to the mRNA pattern observed for proximal tubule markers (Megalin, NaPi-IIa, OAT1) and reflecting the continuous increase in renal cortical structures during development. By contrast, AQPs 2-3-4 showed a later (E15.5) and more abrupt increase, with transient postnatal overexpression. Most AQP genes were expressed earlier and/or stronger in maturing CD-1 kidneys. Furthermore, adult CD-1 kidneys expressed more AQP2 in the collecting ducts, which was reflected by a significant delay in excreting a water load. The expression patterns of proximal vs. distal AQPs and the earlier expression in the CD-1 strain were confirmed by immunoblotting and immunostaining. These data (1) substantiate the clustering of important genes during tubular maturation and (2) demonstrate that genetic variability influences the regulation of the AQP gene family during tubular maturation and water handling by the mature kidney.

Inhibition of nitric oxide synthase reverses permeability changes in a mouse model of acute peritonitis.

Acute peritonitis is the most frequent complication of peritoneal dialysis. Previous studies have suggested a major role for nitric oxide (NO) in the permeability changes and loss of ultrafiltration induced by acute peritonitis. In this study, we further investigated the potential role of NO in a mouse model of peritonitis induced by Escherichia coli Lipopolysaccharide (LPS). A 2-hour peritoneal equilibration test was performed in control and LPS-treated mice using 7% glucose dialysate supplemented or not with the NO synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME). The levels of NO metabolites in the dialysate were maximal 18 hours after LPS injection. At that time, acute peritonitis induced by LPS was reflected by an increased recruitment of leukocytes, an increased intraperitoneal release of interleukin-6, a significant increase in the peritoneal permeability for small solutes, a loss of sodium sieving, and a loss of ultrafiltration in comparison with controls. Addition of L-NAME in LPS-treated mice significantly reversed permeability modifications and prevented the release of NO metabolites into the dialysate. These results confirm that increased NO mediates permeability modifications during acute peritonitis, and illustrate the potential of mouse models to investigate the molecular mechanisms regulating peritoneal permeability.

Functional and molecular characterization of a peritoneal dialysis model in the C57BL/6J mouse.

BACKGROUND: Animal models are important for understanding the physiology and pathophysiology of peritoneal transport during peritoneal dialysis (PD). Mechanistic investigations of rat and rabbit models of PD are mostly based on intervention studies using pharmacologic agents or blocking antibodies. These models may be limited by the time-course, lack of specificity, or side effects of such interventions. Genetically modified mice could provide an attractive alternative to the above models. In this study, we have characterized PD parameters and tested the effect of gender and dialysate volume and/or osmolality in the C57BL/6J mouse. METHODS: Mice were submitted to a 2-hour peritoneal equilibration test in order to obtain permeability parameters. The expression of the water channel aquaporin-1 (AQP1) and endothelial NO synthase (eNOS) was investigated at the protein (immunoblotting, immunostaining) and mRNA [real-time reverse-transcription-polymerase chain reaction (RT-PCR)] levels. The potential effect of gender on these parameters was also studied. RESULTS: Exposure of mice to 2 mL of 3.86% glucose dialysate yielded equilibration curves for urea and glucose, a sodium sieving, and a net ultrafiltration (UF) that were remarkably similar to those obtained in rats. The increase in dialysate volume (from 2 mL to 3 mL and 6 mL) resulted in a higher ultrafiltration and, for the highest volume, an increase in the diffusive mass transport coefficient (MTAC) for urea. The increase in dialysate glucose concentration (from 1.36% to 3.86% and 7%) resulted in increased sodium sieving and higher UF, whereas the MTAC for urea was unchanged. In comparison with males, females had a similar peritoneal transport rate for small solutes but a significantly lower sodium sieving, reflecting a lower AQP1 mRNA and protein expression in the peritoneum. CONCLUSION: These data demonstrate the structural and functional similarity between mouse and rat models of PD, and further emphasize the relevance of mouse models to understand PD in humans. They also suggest that gender may influence water transport and AQP1 expression in the peritoneum.