Lymphangiogenesis and Lymphatic Absorption Are Related and Increased in Chronic Kidney Failure, Independent of Exposure to Dialysis Solutions.

Increased lymphatic absorption might contribute to ultrafiltration failure in peritoneal dialysis (PD). Lymphangiogenesis develops during PD, but little is known about the relationship between its morphologic and functional parameters. The relationships between lymph vessel density, the effective lymphatic absorption rate (ELAR), and fibrosis were investigated in a rat model of chronic kidney failure (CKD) with exposure to dialysis solutions. Wistar rats (n = 44) were allocated to these groups: NKF (normal kidney function), CKD (70% nephrectomy), CKDD [CKD, with daily intraperitoneal (i.p.) Dianeal 3.86% (Baxter Healthcare BV, Utrecht, Netherlands)], CKDP [CKD, with daily i.p. Physioneal 3.86% (Baxter Healthcare BV)]. After 16 weeks, a peritoneal function test was performed, and the ELAR was calculated from the disappearance rate of i.p. dextran 70. The lymph vessel profile density (LVPD) was assessed using STEPanizer image analysis (Java application from Tschanz SA, Bern, Germany) of omental sections after anti-podoplanin immunostaining. Fibrosis was quantified by picro-sirius red staining. The LVPD was significantly increased in CKD rats compared with NKF rats, and no additional effect of dialysis solutions was present. The ELAR was increased in uremic rats compared with NKF rats. For all rats together, the LVPD correlated positively with the ELAR and with the amount of fibrosis. Chronic kidney disease itself induces lymphangiogenesis and fibrosis and increases the ELAR, independent of exposure to dialysis fluids. The ELAR is related to the LVPD in peritoneal tissue.

Hepatic cytochrome P450 deficiency in mouse models for intrahepatic cholestasis predispose to bile salt-induced cholestasis.

Progressive familial intrahepatic cholestasis (PFIC) types 1 and 3 are severe cholestatic liver diseases caused by deficiency of ATB8B1 and ABCB4, respectively. Mouse models for PFIC display mild phenotypes compared with human patients, and this can be explained by the difference in bile salt pool composition. Mice, unlike humans, have the ability to detoxify hydrophobic bile salts by cytochrome P450-mediated (re)hydroxylation and thus have a less toxic bile salt pool. We have crossed mouse models for PFIC1 and PFIC3 with Hrn mice that have a reduced capacity to (re)hydroxylate bile salts. Double transgenes were obtained by backcrossing Atp8b1(G308V/G308V) and Abcb4(-/-) mice with Hrn mice that have a liver-specific disruption of the cytochrome P450 reductase gene and therefore have markedly reduced P450 activity. In these mice, a more hydrophobic bile salt pool was instilled by cholic acid supplementation of the diet, and bile formation and liver pathology was studied. As opposed to single transgenes, Atp8b1(G308V/G308V)/Hrn and Abcb4(-/-)/Hrn mice rapidly developed strong cholestasis that was evidenced by increased plasma bilirubin and bile salt levels. The bile salt pool was more toxic in both models; Atp8b1(G308V/G308V)/Hrn mice had a more hydrophobic plasma pool compared with the single transgene, whereas Abcb4(-/-)/Hrn mice had a more hydrophobic biliary pool compared with the single transgene. In line with these findings, liver damage was not aggravated in Atp8b1(G308V/G308V)/Hrn but was more severe in Abcb4(-/-)/Hrn mice. These data indicate that bile salt pool composition is a critical determinant in the initiation and progression of cholestasis and liver pathology in PFIC1 and PFIC3. Most importantly, our data suggest that the hydrophobicity of the plasma bile salt pool is an important determinant of the severity of cholestasis, whereas the hydrophobicity of the biliary bile salt pool is an important determinant of the severity of liver pathology.

A two-hit approach in the development of an experimental peritoneal sclerosis model.

Models of encapsulating peritoneal sclerosis (EPS) are often based on local administration of chemical irritants. Our aim was to develop a clinically relevant "two-hit" model with incorporation of renal failure and exposure to conventional dialysis solutions. We randomly allocated 36 male Wistar rats that had undergone catheter implantation and a 70% nephrectomy to 3 peritoneal infusion groups. The experimental group was exposed to a 3.86% glucose-based conventional dialysis solution for 8 weeks, after which the animals received a second hit of intraperitoneal blood administration. Two weeks later the rats were humanely euthanized The two control groups were exposed to the conventional dialysis solution alone or to a buffer without glucose for 8 weeks. All animals underwent a peritoneal function test at the end of the experiment. Peritoneal adhesions were counted at autopsy, and omental tissue was obtained for morphometrics. The rats that received blood as a second hit developed numerous intraperitoneal adhesions as seen in EPS, but without cocoon formation. Microscopically, no differences in fibrosis scores and vessel counts were observed between the groups. Peritoneal function parameters were also similar in all groups. The short infusion period could be the reason that we found no differences between the groups, with the exception of the large amount of intraperitoneal adhesions in the experimental group. Modifications to the described rat model are required to develop a clinically relevant EPS model. Besides renal failure and long-term exposure to bioincompatibleperitoneal dialysis solutions, a different second hit or several additional hits could be incorporated into an experimental model of EPS.

Experimental peritoneal sclerosis models should not be based on chlorhexidine gluconate anymore.

BACKGROUND/AIMS: Currently available rodent models of peritoneal sclerosis are not based on clinically relevant factors: renal failure in combination with exposure to bioincompatible fluids. Our aim was to develop a chronic peritoneal infusion model of peritoneal sclerosis in rats with renal failure. METHODS: Male Wistar rats underwent a catheter implantation and a 70% nephrectomy. They were randomly divided into three peritoneal infusion groups: chlorhexidine gluconate/ethanol (CGE) + Dianeal (Baxter Healthcare, Castlebar, Ireland), CGE + buffer (Physioneal without glucose; Baxter, Nivelles, Belgium) and Dianeal alone. After 8 weeks a peritoneal permeability test was performed and omental tissue was obtained for morphometrics. RESULTS: The CGE + Dianeal group (n = 6) and CGE + buffer (n = 6) group showed high peritoneal clearances of small solutes and proteins, ultrafiltration failure, impaired free water transport, severe fibrosis and high vessel counts, but the groups did not differ significantly. The Dianeal group (n = 6) showed significantly lower clearances of small solutes and proteins, normal ultrafiltration and sodium sieving, and significantly lower fibrosis scores and vessel counts. CONCLUSIONS: Abnormalities seen in peritoneal sclerosis can be induced in a peritoneal infusion model in rats with renal failure. However, the addition of a bioincompatible dialysis solution had no contributing role, probably because the effects were overruled by those of CGE.

Addition of a nitric oxide inhibitor to a more biocompatible peritoneal dialysis solution in a rat model of chronic renal failure.

Biocompatible dialysis solutions have been developed to preserve peritoneal membrane morphology and function. Compared with a conventional solution, a combination of glycerol, amino acids, and dextrose in a bicarbonate/lactate buffer (GLAD) led to less peritoneal fibrosis and fewer vessels in a chronic peritoneal exposure model in the rat. However, no concomitant reduction in small-solute transport was observed. We hypothesized that this result could be attributable to peritoneal vasodilation induced by vasoactive substances such as nitric oxide. The aim of the present study was to investigate whether fast transport of small solutes and proteins induced by exposure to GLAD could be influenced by Ngamma -methyl-L-arginine acetate (L-NMMA), an inhibitor of NO. These investigations used our rat model of long-term peritoneal exposure with chronic renal failure. All rats underwent peritoneal catheter implantation and a 70% nephrectomy. Thereafter, the rats were allocated to 3 groups: 16 weeks of peritoneal exposure to GLAD and L-NMMA, to GLAD only, or to buffer (bicarbonate/lactate without any osmotic agent). Afterward, a standard peritoneal permeability analysis adjusted for the rat was performed. Subsequently, the rats were euthanized, and tissue samples were obtained for morphometric determinations. No effect of L-MNNA on the transport of small solutes and proteins was found. Also, no effect on morphology was found. Our findings make it unlikely that NO is directly involved, being more in favor of a direct effect of amino acids on peritoneal transport.

Karl d. Nolph state of the art lecture: feasible and future options for salvation of the peritoneal membrane.

A review is given of the various available strategies that can be used to protect the peritoneal membrane. A discussion of experimental studies on approaches that are still experimental, but that might be applied in patients in the future, follows. The currently available approaches include dietary sodium restriction, use of high-dose loop diuretics and of inhibitors of the renin-angiotensin system. All should preferably be combined with a dialysis prescription aimed at reducing the patient's exposure to glucose and its degradation products. The experimental studies indicate favorable effects of combining osmotic agents, together with drugs that interfere with the polyol pathway and the formation of advanced glycosylation end-products.

Identification of Gene Transcripts Implicated in Peritoneal Membrane Alterations.

♦ BACKGROUND: Permanent stimulation of the peritoneum during peritoneal dialysis (PD) is likely to result in increased expression of genes encoding proteins involved in inflammation and tissue remodeling. Peritoneal fibrosis and neoangiogenesis may develop. ♦ OBJECTIVE: To assess highly expressed genes potentially in volved in peritoneal alterations during PD treatment using an animal model. ♦ METHODS: A PD catheter was implanted in 36 male Wistar rats after 70% nephrectomy. The rats were divided into 3 groups, exposed to dialysis solution for 8 weeks, and sacrificed 2 weeks later. Group B was exposed to a buffer, group D was exposed to a 3.86% glucose-based dialysis solution, and in group D+H, a second hit of intraperitoneal blood on top of the dialysis solution was given to induce the development of peritoneal sclerosis. Before sacrifice, peritoneal function was assessed. Omental tissue was obtained for analysis of gene expression using RT-qPCR. ♦ RESULTS: Fibrosis scores, vessel counts, and peritoneal function parameters were not different between the groups. Genes involved in the transforming growth factor beta signaling pathway, cell proliferation, angiogenesis, and inflammation were more expressed (p < 0.05) in the D+H group. Almost no differences were found between the control groups. We identified 4 genes that were related to peritoneal transport. ♦ CONCLUSION: Already a mid-term peritoneal exposure, when no microscopical and functional alterations are present, provokes activation of gene pathways of cell proliferation, fibrosis, neoangiogenesis, and inflammation.

New Insights into the Effects of Chronic Kidney Failure and Dialysate Exposure on the Peritoneum.

♦ INTRODUCTION: Chronic uremia and the exposure to dialysis solutions during peritoneal dialysis (PD) induce peritoneal alterations. Using a long-term peritoneal exposure model, we compared the effects of chronic kidney failure (CKD) itself and exposure to either a 'conventional' or a 'biocompatible' dialysis solution on peritoneal morphology and function. ♦ METHODS: Wistar rats (Harlan, Zeist, the Netherlands) were grouped into: normal kidney function (NKF), CKD induced by 70% nephrectomy, CKD receiving daily peritoneal infusions with 3.86% glucose Dianeal (CKDD), or Physioneal (both solutions from Baxter Healthcare, Castlebar, Ireland) (CKDP). At 16 weeks, a peritoneal function test was performed, and histology, ultrastructure, and hydroxyproline content of peritoneal tissue were assessed. ♦ RESULTS: Comparing CKD with NKF, peritoneal transport rates were higher, mesothelial cells (MC) displayed increased number of microvilli, blood and lymph vasculature expanded, vascular basal lamina appeared thicker, with limited areas of duplication, and fibrosis had developed. All alterations, except lymphangiogenesis, were enhanced by exposure to both dialysis fluids. Distinct MC alterations were observed in CKDD and CKDP, the latter displaying prominent basolateral protrusions. In addition, CKDP was associated with a trend towards less fibrosis compared to CKDD. ♦ CONCLUSIONS: Chronic kidney failure itself induced peritoneal alterations, which were in part augmented by exposure to glucose-based dialysis solutions. Overall, the conventional and biocompatible solutions had similar long-term effects on the peritoneum. Importantly, the latter may attenuate the development of fibrosis.

The effects of a dialysis solution with a combination of glycerol/amino acids/dextrose on the peritoneal membrane in chronic renal failure.

BACKGROUND: Long-term peritoneal dialysis (PD) with conventional glucose based, lactate-buffered PD fluids may lead to morphological and functional alterations of the peritoneal membrane. It was hypothesized that long-term exposure to a different buffer and a mixture of osmotic agents would cause less peritoneal abnormality. OBJECTIVES: To investigate the effects of long-term exposure to a bicarbonate/lactate-buffered dialysis solution with a mixture of osmotic agents: glycerol 1.4%, amino acids 0.5%, and dextrose 1.1% (= 1% glucose) (GLAD) in a rat model with chronic kidney failure. METHODS: All rats underwent a peritoneal catheter implantation and a 70% nephrectomy. Thereafter, the rats were randomly divided into 3 groups: GLAD, 3.86% Dianeal (Baxter, Nivelles, Belgium), and buffer (Physioneal without glucose, Baxter). All rats were infused daily for 16 weeks with the appropriate PD fluid. Afterwards, a peritoneal permeability analysis (SPARa) was performed using 3.86% Physioneal in all groups. After the SPARa, the rats were sacrificed to obtain tissue samples for morphometric determinations. Omental tissue was stained with picro Sirius red for assessment of fibrosis and with CD31 for vessel density. RESULTS: GLAD and Dianeal showed faster small solute transport compared to the hypotonic buffer. No differences between the groups were present in ultrafiltration. Dianeal had the lowest value for free water transport and the highest protein clearances. Total triglyceride in plasma was not different between GLAD and the buffer. Vessel density after GLAD exposure (20 V/F) was very similar to the value found for the buffer solution (17 V/F); Dianeal caused a significantly higher value (35 V/F, p < 0.01). Also, the amount of fibrosis was higher in the Dianeal-exposed rats (p < 0.01). CONCLUSION: Both hypertonic dialysis solutions increased peritoneal solute transport. GLAD exposure was associated with the best preservation of peritoneal morphology. The results of GLAD were very similar to those of the bicarbonate/lactate-buffered solution without osmotic agents. Studies in humans are needed for further assessment of GLAD.

CRF1 not glucocorticoid receptors mediate prepulse inhibition deficits in mice overexpressing CRF.

BACKGROUND: Both corticotropin-releasing factor (CRF) and glucocorticoid receptors (GR) are implicated in the psychotic symptoms of psychiatric disorders. Correspondingly, it is of interest to determine their respective involvement in the sensorimotor gating deficits displayed by transgenic mice overexpressing CRF. These mice reveal lifelong elevations of CRF and corticosterone levels. METHODS: Effects of the GR antagonists ORG34517 (5-45 mg/kg by mouth [PO]) and mifepristone (5-45 mg/kg PO) and the CRF(1) receptor antagonists CP154,526 (20-80 mg/kg intraperitoneally [IP]) and DMP695 (2.5-40.0 mg/kg IP) on prepulse inhibition (PPI) of the acoustic startle response were studied in mice overexpressing CRF and in their wild-type littermates. In addition, PPI was measured in both genotypes 2 weeks after adrenalectomy with or without exogenous corticosterone administration via subcutaneous pellet implant (20 mg corticosterone). RESULTS: ORG34517 and mifepristone did not influence perturbation of PPI in mice overexpressing CRF; reducing corticosterone levels by adrenalectomy likewise did not improve PPI. Further, elevation in corticosterone levels by pellet implantation did not disrupt PPI in wild-type mice. Conversely, both CRF(1) receptor antagonists, CP154,526 (40-80 mg/kg IP) and DMP695 (40 mg/kg IP), significantly restored PPI in CRF-overexpressing mice. CONCLUSIONS: Sustained overactivation of CRF(1) receptors rather than excessive GR receptor stimulation underlies impaired sensorimotor gating in CRF-overexpressing mice. CRF(1) receptors thus may play a role in the expression of psychotic features in stress-related psychiatric disorders.