L-arginine:glycine amidinotransferase deficiency protects from metabolic syndrome.

Phosphorylated creatine (Cr) serves as an energy buffer for ATP replenishment in organs with highly fluctuating energy demand. The central role of Cr in the brain and muscle is emphasized by severe neurometabolic disorders caused by Cr deficiency. Common symptoms of inborn errors of creatine synthesis or distribution include mental retardation and muscular weakness. Human mutations in l-arginine:glycine amidinotransferase (AGAT), the first enzyme of Cr synthesis, lead to severely reduced Cr and guanidinoacetate (GuA) levels. Here, we report the generation and metabolic characterization of AGAT-deficient mice that are devoid of Cr and its precursor GuA. AGAT-deficient mice exhibited decreased fat deposition, attenuated gluconeogenesis, reduced cholesterol levels and enhanced glucose tolerance. Furthermore, Cr deficiency completely protected from the development of metabolic syndrome caused by diet-induced obesity. Biochemical analyses revealed the chronic Cr-dependent activation of AMP-activated protein kinase (AMPK), which stimulates catabolic pathways in metabolically relevant tissues such as the brain, skeletal muscle, adipose tissue and liver, suggesting a mechanism underlying the metabolic phenotype. In summary, our results show marked metabolic effects of Cr deficiency via the chronic activation of AMPK in a first animal model of AGAT deficiency. In addition to insights into metabolic changes in Cr deficiency syndromes, our genetic model reveals a novel mechanism as a potential treatment option for obesity and type 2 diabetes mellitus.

Homoarginine levels are regulated by L-arginine:glycine amidinotransferase and affect stroke outcome: results from human and murine studies.

BACKGROUND: Endogenous arginine homologues, including homoarginine, have been identified as novel biomarkers for cardiovascular disease and outcomes. Our studies of human cohorts and a confirmatory murine model associated the arginine homologue homoarginine and its metabolism with stroke pathology and outcome. METHODS AND RESULTS: Increasing homoarginine levels were independently associated with a reduction in all-cause mortality in patients with ischemic stroke (7.4 years of follow-up; hazard ratio for 1-SD homoarginine, 0.79 [95% confidence interval, 0.64-0.96]; P=0.019; n=389). Homoarginine was also independently associated with the National Institutes of Health Stroke Scale+age score and 30-day mortality after ischemic stroke (P<0.05; n=137). A genome-wide association study revealed that plasma homoarginine was strongly associated with single nucleotide polymorphisms in the L-arginine:glycine amidinotransferase (AGAT) gene (P<2.1 × 10(-8); n=2806), and increased AGAT expression in a cell model was associated with increased homoarginine. Next, we used 2 genetic murine models to investigate the link between plasma homoarginine and outcome after experimental ischemic stroke: (1) an AGAT deletion (AGAT(-/-)) and (2) a guanidinoacetate N-methyltransferase deletion (GAMT(-/-)) causing AGAT upregulation. As suggested by the genome-wide association study, homoarginine was absent in AGAT(-/-) mice and increased in GAMT(-/-) mice. Cerebral damage and neurological deficits in experimental stroke were increased in AGAT(-/-) mice and attenuated by homoarginine supplementation, whereas infarct size in GAMT(-/-) mice was decreased compared with controls. CONCLUSIONS: Low homoarginine appears to be related to poor outcome after ischemic stroke. Further validation in future trials may lead to therapeutic adjustments of homoarginine metabolism that alleviate stroke and other vascular disorders.

Lethal phenotype in conditional late-onset arginase 1 deficiency in the mouse.

Human arginase deficiency is characterized by hyperargininemia and infrequent episodes of hyperammonemia, which lead to neurological impairment with spasticity, loss of ambulation, seizures, and severe mental and growth retardation; uncommonly, patients suffer early death from this disorder. In a murine targeted knockout model, onset of the phenotypic abnormality is heralded by weight loss at around day 15, and death occurs typically by postnatal day 17 with hyperargininemia and markedly elevated ammonia. This discrepancy between the more attenuated juvenile-onset human disease and the lethal neonatal murine model has remained suboptimal for studying and developing therapy for the more common presentation of arginase deficiency. These investigations aimed to address this issue by creating an adult conditional knockout mouse to determine whether later onset of arginase deficiency also resulted in lethality. Animal survival and ammonia levels, body weight, circulating amino acids, and tissue arginase levels were examined as outcome parameters after widespread Cre-recombinase activation in a conditional knockout model of arginase 1 deficiency. One hundred percent of adult female and 70% of adult male mice died an average of 21.0 and 21.6 days, respectively, after the initiation of tamoxifen administration. Animals demonstrated elevated circulating ammonia and arginine at the onset of phenotypic abnormalities. In addition, brain and liver amino acids demonstrated abnormalities. These studies demonstrate that (a) the absence of arginase in adult animals results in a disease profile (leading to death) similar to that of the targeted knockout and (b) the phenotypic abnormalities seen in the juvenile-onset model are not exclusive to the age of the animal but instead to the biochemistry of the disorder. This adult model will be useful for developing gene- and cell-based therapies for this disorder that will not be limited by the small animal size of neonatal therapy and for developing a better understanding of the characteristics of hyperargininemia.

Guanidino compound levels in blood, cerebrospinal fluid, and post-mortem brain material of patients with argininemia.

The paucity of hyperammonemic crises together with spasticity, only seen in human arginase I deficient patients and not in patients with other urea cycle disorders, forces a search for candidates other than ammonia to associate with the pathophysiology and symptomatology. Therefore, we determined arginine together with some catabolites of arginine in blood and cerebrospinal fluid of these patients as well as in extremely rare post-mortem brain material of two patients with argininemia. The levels of alpha-keto-delta-guanidinovaleric acid, argininic acid and alpha-N-acetylarginine correlate with the arginine levels in blood and cerebrospinal fluid of patients with imposed or spontaneous protein restriction. The levels in blood are higher than the upper limit of normal in all studied patients. In addition to the highly increased levels of these same compounds in blood of a child with argininemia, the increase of guanidinoacetic acid, 24h before death, is remarkable. However, the manifest increases of these studied catabolites of arginine are not seen in post-mortem brain material of the same pediatric patient. Otherwise a clear increase of guanidinoacetic acid in post-mortem brain material of an adult patient was shown. A similar, comparable increase of homoarginine in both studied post-mortem brain materials is observed. Therefore the study of the pathobiochemistry of arginine in argininemia must be completed in the future by the determination of the end catabolites of the nitric oxide and agmatine biosynthesis pathways in the knockouts as well as in the patients to evaluate their role, together with the here studied catabolites, as candidates for association with pathophysiology and symptomatology.

Dimethylarginine levels in cerebrospinal fluid of hyperacute ischemic stroke patients are associated with stroke severity.

We hypothesise that asymmetric and symmetric dimethylarginine (ADMA, SDMA) are released in cerebrospinal fluid (CSF) due to ischemia-induced proteolysis and that CSF dimethylarginines are related to stroke severity. ADMA and SDMA were measured in CSF of 88 patients with ischemic stroke or TIA within 24 h after stroke onset (mean 8.6 h) and in 24 controls. Stroke severity was assessed by the National Institutes of Health Stroke Scale (NIHSS) score at admission. Outcome was evaluated by institutionalization due to stroke and the modified Rankin scale. Dimethylarginine levels were higher in patients with stroke than in TIA patients, who had higher levels than controls and correlated with the NIHSS. Logistic regression analysis confirmed that dimethylarginines were independently associated with stroke severity. The SDMA/ADMA ratio did not differ significantly between controls and stroke patients. CSF dimethylarginine levels are increased in hyperacute ischemic stroke and are associated with stroke severity.

Impact of increasing haemodialysis frequency versus haemodialysis duration on removal of urea and guanidino compounds: a kinetic analysis.

BACKGROUND: Patients with renal failure retain a large variety of uraemic solutes, characterized by different kinetic behaviour. It is not entirely clear what the impact is of increasing dialysis frequency and/or duration on removal efficiency, nor whether this impact is the same for all types of solutes. METHODS: This study was based on two-compartmental kinetic data obtained in stable haemodialysis patients (n = 7) for urea, creatinine (CREA), guanidinosuccinic acid (GSA) and methylguanidine (MG). For each individual patient, mathematical simulations were performed for different dialysis schedules, varying in frequency, duration and intensity. For each dialysis schedule, plasmatic and extraplasmatic weekly time-averaged concentrations (TAC) were calculated, as well as their %difference to weekly TAC of the reference dialysis schedule (three times weekly 4 h). RESULTS: Increasing dialysis duration was most beneficial for CREA and MG, which are distributed in a larger volume (54.0 +/- 5.9 L and 102.6 +/- 33.9 L) than urea (42.7 +/- 6.0 L) [plasmatic weekly TAC decrease of 31.5 +/- 3.2% and 31.8 +/- 3.8% for CREA and MG with Q(B) of 200 mL/min, compared to 25.7 +/- 3.2% for urea (P = 0.001 and P < 0.001)]. Increasing dialysis frequency resulted only in a limited increase in efficiency, most pronounced for solutes distributed in a small volume like GSA (30.6 +/- 4.2 L). Increasing both duration and frequency results in weekly TAC decreases of >65% for all solutes. Comparable results were found in the extraplasmatic compartment. CONCLUSION: Prolonged dialysis significantly reduces solute concentration levels, especially for those solutes that are distributed in a larger volume. Increasing both dialysis frequency and duration is the superior dialysis schedule.

Increased plasma and tissue guanidino compounds in a mouse model of hyperargininemia.

In humans, arginase I (AI)-deficiency results in hyperargininemia, a metabolic disorder with symptoms of progressive neurological and intellectual impairment, spasticity, persistent growth retardation, and episodic hyperammonemia. A deficiency of arginase II (AII) has never been detected and the clinical disorder, if any, associated with its deficiency has not been defined. Since the spasticity and paucity of hyperammonemic crises seen in human AI-deficient patients are not features of the other urea cycle disorders, the likelihood of ammonia as the main neuropathogenic agent becomes extremely low, and the modest elevations of arginine seen in the brains of our mouse model of hyperargininemia make it an unlikely candidate as well. Specific guanidino compounds, direct or indirect metabolites of arginine, are elevated in the blood of patients with uremia. Other guanidino compounds are also increased in plasma and cerebrospinal fluid of hyperargininemic patients making them plausible as neurotoxins in these disorders. We analyzed several guanidino compounds in our arginase single and double knockout animals and found that alpha-keto-delta-guanidinovaleric acid, alpha-N-acetylarginine, and argininic acid were increased in the brain tissue from the AI knockout and double knockout animals. Several compounds were also increased in the plasma, liver, and kidneys. This is the first time that several of the guanidino compounds have been shown to be elevated in the brain tissue of an arginase-deficient mammal, and it further supports their possible role as the neuropathogenic agents responsible for the complications seen in arginase deficiency.

Accumulation of methylguanidine and changes in guanidino compound levels in plasma, urine, and kidneys of furosemide-treated rats.

Antidiuresis and renal diseases alter the levels of guanidino compounds (GCs) in various tissues. Therefore, we hypothesized that diuresis could also disturb GC metabolism, storage, and elimination. In this study, rats were made diuretic to analyze GC levels in plasma, urine, and kidneys. Furosemide was chosen because of its wide use in various human pathologies. Rats were injected intraperitoneally 5 or 10 mg furosemide spread over a 24-hour cycle. Urine was collected over a period of 24 hours before and during furosemide treatment. Plasma was obtained from arterial blood. Renal zones were dissected. The GCs were determined by liquid chromatography. Five milligrams of furosemide provoked a significant increase in plasma and urine levels of GCs compared with those of the controls. The renal distribution and content of GCs were weakly modified by furosemide except for methylguanidine (MG). The level of MG was enhanced by 10 to 16 times in all renal zones. The MG level was 60% higher in renal zones of rats treated with 10 rather than 5 mg furosemide. The fractional excretion of MG was decreased by furosemide. Our data suggest that MG accumulation in kidney and plasma was caused by furosemide, which might induce MG synthesis, and that MG washout from tissue cells into urine by furosemide through the kidney may cause an increase in MG in the kidney.

Guanidino compounds after creatine supplementation in renal failure patients and their relation to inflammatory status.

BACKGROUND: Specific guanidino compounds have been described as uraemic toxins and their concentrations are increased in renal failure due to dimished glomerular filtration, whereas the guanidino compound creatine is used as a performance-enhancing substance in athletes. The present study investigates the effects of creatine supplementation on plasma guanidino compounds in a chronic haemodialysis population. METHODS: Twenty male haemodialysis patients were included in a placebo-controlled cross-over trial. Patients were treated with creatine (2 g/day) or placebo during two treatment periods of 4 weeks, separated by a washout of 4 weeks. Plasma guanidino compounds and routine biochemical parameters were determined, as well as the prognostic inflammatory and nutritional index (PINI). RESULTS: Upon creatine supplementation, guanidinoacetate concentrations decreased by 15%, due to inhibition of creatine synthesis. Concentrations of alpha-keto-delta-guanidinovaleric acid increased three-fold and argininic acid concentrations doubled. Guanidinosuccinate concentrations did not change, but correlated inversely with CRP (r = -0.736; P = 0.001), PINI-score (r = -0.716; P = 0.002) and correlated positively with plasma urea concentration (r = 0.54; P = 0.02). CONCLUSIONS: Creatine supplementation in haemodialysis patients significantly altered the concentration of specific guanidino compounds. Guanidinosuccinate correlated positively with plasma urea and negatively with inflammation markers.

Complex compartmental behavior of small water-soluble uremic retention solutes: evaluation by direct measurements in plasma and erythrocytes.

BACKGROUND: Although scanty data suggest that large solutes show kinetic behavior different from urea, there are virtually no data comparing the kinetics of urea with those of other small water-soluble uremic compounds, which are believed to behave similarly. STUDY DESIGN: Cross-sectional study of kinetics of urea and guanidino compounds in plasma and erythrocyte compartments during a single hemodialysis session. SETTING & PARTICIPANTS: Six stable hemodialysis patients on standard low-flux dialysis therapy. PREDICTORS: Reduction ratios (RRs) of urea calculated from plasma and erythrocyte concentrations. OUTCOMES: RRs for guanidino compounds calculated from measurements of both plasma and erythrocyte concentrations. MEASUREMENTS: Blood samples were collected from the dialyzer inlet and outlet at 0, 5, 15, 30, and 120 minutes and at the end of the session. Plasma and erythrocyte concentrations of urea and guanidino compounds (creatinine [CTN], guanidinosuccinic acid [GSA], guanidinoacetic acid [GAA], guanidine [G], and methylguanidine [MG]) were determined. RESULTS: Postdialysis plasma RR was higher for GSA (82% +/- 3%) compared with urea (77% +/- 2%; P < 0.01), whereas CTN (69% +/- 4%), GAA (49% +/- 14%), G (55% +/- 7%), and MG (55% +/- 7%) showed smaller RRs (P < 0.01). In erythrocytes, GSA (45% +/- 1%), G (10% +/- 13%), and MG (27% +/- 10%) showed markedly smaller RRs than urea (59% +/- 6%; P < 0.05). Finally, significant differences were found between plasma and erythrocyte RRs for urea, GSA, G, and MG (P < 0.01). LIMITATIONS: Discrepancies were found between the biochemical and mathematical approaches. Hence, the erythrocyte compartment does not necessarily conform to the kinetic nonperfused compartment. CONCLUSIONS: Our data indicate by means of direct estimations that the compartmental behaviors of guanidino compounds and urea are substantially different. Hence, we should consider that not all changes in concentrations in uremia and dialysis are representatively reflected by urea kinetics, even when considering other small water-soluble substances, such as the guanidino compounds.

Analysis of cholinergic markers, biogenic amines, and amino acids in the CNS of two APP overexpression mouse models.

Two transgenic mouse models expressing mutated human amyloid precursor protein and previously found to display cognitive and behavioural alterations, reminiscent of Alzheimer patients' symptomatology, were scrutinised for putative brain region-specific changes in neurochemical parameters. Brains of NSE-hAPP751m-57, APP23 and wild-type mice were microdissected to perform brain region-specific neurochemical analyses. Impairment of cholinergic transmission, the prominent neurochemical deficit in Alzheimer brain, was examined; acetylcholinesterase and choline acetyltransferase activity levels were determined as markers of the cholinergic system. Since Alzheimer neurodegeneration is not restricted to the cholinergic system, brain levels of biogenic amines and metabolites, and amino acidergic neurotransmitters and systemic amino acids were analysed as well. Cholinergic dysfunction, reflected in reduced enzymatic activity in the basal forebrain nuclei, was restricted to the APP23 model, which also exhibited more outspoken and more widespread changes in other neurotransmitter systems. Significant changes in compounds of the noradrenergic and serotonergic system were observed, as well as alterations in levels of the inhibitory neurotransmitter glycine and systemic amino acids. These observations were clearly in occurrence with the more pronounced histopathological and behavioural phenotype of the APP23 model. As transgenic models often do not represent an end-stage of the disease, some discrepancies with results from post-mortem human Alzheimer brain analyses were apparent; in particular, no significant alterations in excitatory amino acid levels were detected. Our findings of brain region-specific alterations in compound levels indicate disturbed neurotransmission pathways, and greatly add to the validity of APP23 mice as a model for Alzheimer's disease. Transgenic mouse models may be employed as a tool to study early-stage neurochemical changes, which are often not accessible in Alzheimer brain.

Biochemical and behavioural phenotyping of a mouse model for GAMT deficiency.

Deficiency of guanidinoacetate N-methyltransferase (GAMT) is the first described creatine (CT) deficiency syndrome in man, biochemically characterized by accumulation of guanidinoacetic acid (GAA) and depletion of CT. Patients exhibit severe developmental and muscular problems. We created a mouse model for GAMT deficiency, which exerts biochemical changes comparable with those found in human GAMT-deficient subjects. CT and creatinine (CTN) levels are significantly decreased and GAA is increased in knockout (KO) mice. In patients, other guanidino compounds (GCs) appear to be altered as well, which may also contribute to the symptomatology. Extensive evaluation of GCs levels in the GAMT mouse model was therefore considered appropriate. Concentrations of 13 GCs in plasma, 24-h urine, brain and muscle of GAMT mice were measured. We also report on the detailed behavioural characterization of this model for GAMT deficiency. Besides an increase of GAA and a decrease of CT and CTN in plasma, 24-h urine, brain and muscle of KO mice, we observed a significant increase of other GCs in brain and muscle that was sometimes reflected in plasma and/or urine. KO mice displayed mild cognitive impairment. In general, it could be concluded that the GAMT mouse model is very useful for biochemical research of GAMT deficiency, but shows only a mild cognitive deficit.

GSA: behavioral, histological, electrophysiological and neurochemical effects.

Renal insufficient patients suffer from a variety of complications as direct and indirect consequence of accumulation of retention solutes. Guanidinosuccinic acid (GSA) is an important probable uremic toxin, increased in plasma, urine, cerebrospinal fluid and brain of patients with uremia and supposed to play a role in the pathogenesis of some neurological symptoms. GSA, an NMDA-receptor agonist and GABA-receptor antagonist, is suggested to act as an excitotoxin and shown to be convulsive. The effect of hippocampal (i.h.) GSA injection on behavior and hippocampal volume in mice is presented here. In addition, hippocampal cGMP concentration after systemic injection of GSA was measured. The effect of co-application of NMDA-receptor antagonist CGP37849 with GSA was tested, in vivo, after hippocampal GSA injection and, in vitro, on GSA evoked currents in spinal cord neurons. A significant dose-dependent effect of i.h. injection of GSA on cognitive performance, activity and social exploratory behavior was observed. There was a protective effect of CGP37849 on GSA induced behavioral alterations. Volume of hippocampal cornu ammonis region decreased significantly and dose-dependently after GSA injection. Systemic GSA injection increased cGMP concentration in hippocampal formation. It can be concluded that GSA is an important neurotoxin. As GSA is increased in patients with uremia, it probably contributes to their neurological symptoms. Knowledge of neurotoxic effects and mechanisms of action of GSA and other uremic retention solutes could help in the development of more efficient treatment of uremic patients. Animal models like the 'GSA mouse model' are useful tools for research in this context.

Overexpression of arginase I in enterocytes of transgenic mice elicits a selective arginine deficiency and affects skin, muscle, and lymphoid development.

BACKGROUND: Arginine is required for the detoxification of ammonia and the synthesis of proteins, nitric oxide, agmatine, creatine, and polyamines, and it may promote lymphocyte function. In suckling mammals, arginine is synthesized in the enterocytes of the small intestine, but this capacity is lost after weaning. OBJECTIVE: We investigated the significance of intestinal arginine production for neonatal development in a murine model of chronic arginine deficiency. DESIGN: Two lines of transgenic mice that express different levels of arginase I in their enterocytes were analyzed. RESULTS: Both lines suffer from a selective but quantitatively different reduction in circulating arginine concentration. The degree of arginine deficiency correlated with the degree of retardation of hair and muscle growth and with the development of the lymphoid tissue, in particular Peyer's patches. Expression of arginase in all enterocytes was necessary to elicit this phenotype. Phenotypic abnormalities were reversed by daily injections of arginine but not of creatine. The expression level of the very arginine-rich skin protein trichohyalin was not affected in transgenic mice. Finally, nitric oxide synthase-deficient mice did not show any of the features of arginine deficiency. CONCLUSIONS: Enterocytes are important for maintaining arginine homeostasis in neonatal mice. Graded arginine deficiency causes graded impairment of skin, muscle, and lymphoid development. The effects of arginine deficiency are not mediated by impaired synthesis of creatine or by incomplete charging of arginyl-transfer RNA.

Plasma guanidino compounds are altered by oral creatine supplementation in healthy humans.

Although creatine is one of the most widely used nutritional supplements for athletes as well as for patients with neuromuscular disorders, the effects of oral creatine supplementation on endogenous creatine synthesis in humans remains largely unexplored. The aim of the present study was to investigate the metabolic consequences of a frequently used, long-term creatine ingestion protocol on the circulating creatine synthesis precursor molecules, guanidinoacetate and arginine, and their related guanidino compounds. For this purpose, 16 healthy young volunteers were randomly divided to ingest in a double-blind fashion either creatine monohydrate or placebo (maltodextrine) at a dosage of 20 g/day for the first week (loading phase) and 5 g/day for 19 subsequent wk (maintenance phase). Fasting plasma samples were taken at baseline and at 1, 10, and 20 wk of supplementation, and guanidino compounds were determined. Plasma guanidinoacetate levels were reduced by 50% after creatine loading and remained approximately 30% reduced throughout the maintenance phase. Several circulating guanidino compound levels were significantly altered after creatine loading but not during the maintenance phase: homoarginine (+35%), alpha-keto-delta-guanidinovaleric acid (+45%), and argininic acid (+75%) were increased, whereas guanidinosuccinate was reduced (-25%). The decrease in circulating guanidinoacetate levels suggests that exogenous supply of creatine chronically inhibits endogenous synthesis at the transamidinase step in humans, supporting earlier animal studies showing a powerful repressive effect of creatine on l-arginine:glycine amidinotransferase. Furthermore, these data suggest that this leads to enhanced utilization of arginine as a substrate for secondary pathways.

Homeostatic plasticity of striatal neurons intrinsic excitability following dopamine depletion.

The striatum is the major input structure of basal ganglia and is involved in adaptive control of behaviour through the selection of relevant informations. Dopaminergic neurons that innervate striatum die in Parkinson disease, leading to inefficient adaptive behaviour. Neuronal activity of striatal medium spiny neurons (MSN) is modulated by dopamine receptors. Although dopamine signalling had received substantial attention, consequences of dopamine depletion on MSN intrinsic excitability remain unclear. Here we show, by performing perforated patch clamp recordings on brain slices, that dopamine depletion leads to an increase in MSN intrinsic excitability through the decrease of an inactivating A-type potassium current, I(A). Despite the large decrease in their excitatory synaptic inputs determined by the decreased dendritic spines density and the increase in minimal current to evoke the first EPSP, this increase in intrinsic excitability resulted in an enhanced responsiveness to their remaining synapses, allowing them to fire similarly or more efficiently following input stimulation than in control condition. Therefore, this increase in intrinsic excitability through the regulation of I(A) represents a form of homeostatic plasticity allowing neurons to compensate for perturbations in synaptic transmission and to promote stability in firing. The present observations show that this homeostatic ability to maintain firing rates within functional range also occurs in pathological conditions, allowing stabilizing neural computation within affected neuronal networks.

Kinetic behavior of urea is different from that of other water-soluble compounds: the case of the guanidino compounds.

BACKGROUND: Although patients with renal failure retain a large variety of solutes, urea is virtually the only currently applied marker for adequacy of dialysis. Only a limited number of other compounds have up until now been investigated regarding their intradialytic kinetics. Scant data suggest that large solutes show a kinetic behavior that is different from urea. The question investigated in this study was whether other small water-soluble solutes, such as some guanidino compounds, show a kinetic behavior comparable or dissimilar to that of urea. METHODS: This study included 7 stable conventional hemodialysis patients without native kidney function undergoing low flux polysulphone dialysis (F8 and F10HPS). Blood samples were collected from the inlet and outlet bloodlines immediately before the dialysis session, after 5, 15, 30, 120 minutes, and immediately after discontinuation of the session. Plasma concentrations of urea, creatinine (CTN), creatine (CT), guanidinosuccinic acid (GSA), guanidinoacetic acid (GAA), guanidine (G), and methylguanidine (MG) were used to calculate corresponding dialyzer clearances. A two-pool kinetic model was fitted to the measured plasma concentration profiles, resulting in the calculation of the perfused volume (V(1)), the total distribution volume (V(tot)), and the intercompartmental clearance (K(12)); solute generation and overall ultrafiltration were determined independently. RESULTS: No significant differences were observed between V(1) and K(12) for urea (6.4 +/- 3.3 L and 822 +/- 345 mL/min, respectively) and for the guanidino compounds. However, with respect to V(tot), GSA was distributed in a smaller volume (30.6 +/- 4.2 L) compared to urea (42.7 +/- 6.0L) (P < 0.001), while CTN, CT, GAA, G, and MG showed significantly higher volumes (54.0 +/- 5.9 L, 98.0 +/- 52.3 L, 123.8 +/- 66.9 L, 89.7 +/- 21.4 L, 102.6 +/- 33.9 L, respectively; P= 0.004, = 0.033, = 0.003, < 0.001, = 0.001, respectively). These differences resulted in divergent effective solute removal: 67% (urea), 58% (CTN), 42% (CT), 76% (GSA), 37% (GAA), 43% (G), and 42% (MG). CONCLUSION: The kinetics of the guanidino compounds under study are different from that of urea; hence, urea kinetics are not representative for the removal of other uremic solutes, even if they are small and water-soluble like urea.

Dehydration modifies guanidino compound concentrations in the different zones of the rat kidney.

Guanidino compounds (GCs) related to arginine (Arg) are unevenly distributed along the cortico-papillary axis of the rat kidney. Inasmuch as the concentration of alpha-keto-delta-guanidinovaleric acid (alpha-keto-delta-GVA), guanidinosuccinic acid (GSA), creatinine (CTN), gamma-guanidinobutyric acid (gamma-GBA) and methylguanidine (MG) increased steeply along the inner medulla in parallel to the urea and osmotic gradients, the question arose as to whether dehydration enhances their renal content and distribution. To examine this possibility, adult male rats were dehydrated by removing the drinking water for 24 or 48 h. The kidneys were sliced and cut in seven sections along the cortico-papillary axis. Twelve GCs were determined by liquid chromatography in each renal zone. Dehydration modified GC concentrations and regional distribution. The renal content of Arg, guanidine and MG was decreased while that of alpha-keto-delta-GVA, gamma-GBA, alpha- N-acetyl-arginine and homoarginine remained unchanged. In contrast, GSA, guanidinoacetic acid (GAA), creatine (CT), CTN and beta-guanidinopropionic acid (beta-GPA) concentrations were enhanced significantly in different renal zones after 24 and 48 h dehydration. In addition, the tissue level of GCs supplying energy, such as CT and beta-GPA, the precursor of CT (GAA) and its metabolite (CTN) were enhanced under dehydration. Arg and CT account for 80-90% of the GCs located in the renal cortex. Variations of some GC levels under dehydration may modify enzyme activities, renal metabolism and cell function.

Nutritional effects of increasing dialysis dose by adding an icodextrin daytime dwell to Nocturnal Intermittent Peritoneal Dialysis (NIPD) in children.

BACKGROUND: To assess the need to adapt dietary prescriptions, we studied potential effects of increasing the dialysis dose by adding a daytime icodextrin dwell, in children on Nocturnal Intermittent Peritoneal Dialysis (NIPD), on peritoneal amino acids (AA) and albumin loss, AA, albumin, cholesterol and fibrinogen plasma levels and nutritional intake. METHODS: A cross-over study in eight children (age 2-12 years) on NIPD at baseline (week 1). INTERVENTION: to increase dialysis dose we added a daytime dwell with 1100 ml/m(2) icodextrin solution for a week (week 2). MAIN OUTCOME MEASURES: peritoneal albumin loss (quantified by nephelometry) and AA loss (quantified by liquid chromatography mass spectrometry) in the last 72 h dialysate collections of weeks 1 and 2. On days 7 and 14, morning blood sample was taken for urea, creatinine, plasma AA levels, serum albumin, cholesterol and fibrinogen determination. Nutritional intake diaries were kept throughout the study period. RESULTS: Weekly dialysis creatinine clearance increased from 35 to 65 l/1.73 m(2) (P<0.0001) and Kt/V from 1.99 to 2.54 (P<0.01). Peritoneal albumin loss did not change significantly (2.4+/-0.4 to 2.4+/-0.3 g/m(2)/24 h) nor did serum albumin (3.25+/-0.52 to 3.21+/-0.25 g/dl), cholesterol (216+/-73 to 240+/-61 mg/dl) and fibrinogen (385+/-40 to 436+/-64 mg/dl). There was a significant increase in loss of essential (EAA) [1122+/-200 to 2104+/-417 mg/m(2)/week (P<0.0001)] and non-essential amino acids (NEAA) [6160+/-1341 to 10406+/-2899 mg/m(2)/week (P<0.001)]. Plasma AA levels did not change significantly except for a drop in histidine and glutamine. Dietary protein intake did not change from 43+/-12 to 41+/-8 g/m(2)/day, caloric intake from 73+/-21 to 70+/-24 kcal/kg/day. CONCLUSIONS: Increasing dialysis dose by introducing a daytime icodextrin dwell during a week does not affect peritoneal albumin loss, serum albumin, cholesterol and fibrinogen levels nor dietary intake on a short term. There is a significant increase in EAA and NEAA loss without change in plasma levels. We suggest monitoring dietary intake when adding a daytime icodextrin dwell in children.

Involvement of voltage- and ligand-gated Ca2+ channels in the neuroexcitatory and synergistic effects of putative uremic neurotoxins.

BACKGROUND: Renal failure has been viewed as a state of cellular calcium toxicity due to the retention of small fast-acting molecules. We have tested this hypothesis and identified potentially neuroexcitatory compounds among a number of putative uremic neurotoxins by examining the acute in vitro effects of these compounds on cultured central neurons. The in vitro neuroexcitatory and synergistic effects of guanidinosuccinate and spermine were also examined in vivo. METHODS: The acute effects of 17 candidate uremic neurotoxins on murine spinal cord neurons in primary dissociated cell culture were investigated using the tight-seal whole-cell recording technique. The compounds studied comprised low-molecular-weight solutes like urea, indoles, guanidino compounds, polyamines, purines and phenoles, homocysteine, orotate, and myoinositol. Currents evoked by these compounds were further examined using various ligand- and voltage-gated ion channel blockers. The acute in vivo effects of guanidinosuccinate and spermine were behaviorally assessed following their injection in mice. RESULTS: It was shown that 3-indoxyl sulfate, guanidinosuccinate, spermine, and phenol evoked significant whole-cell currents. Inward whole-cell current evoked by 3-indoxyl sulfate was not blocked by any of the applied ligand- or voltage-gated ion channel blockers, and the compound appeared to influence miscellaneous membrane ionic conductances, probably involving voltage-gated Ca2+ channels as well. Phenol-evoked outward whole-cell currents were at least partly due to the activation of voltage-gated K+ channels, but may also involve a variety of other ionic conductances. On the other hand, inward whole-cell currents evoked by guanidinosuccinate and spermine were shown to be due to specific interaction with voltage- and ligand-gated Ca2+ channels. Guanidinosuccinate-evoked current was caused by activation of N-methyl-d-aspartate (NMDA) receptor-associated ion channels. Low (micromol/L) concentrations of spermine potentiated guanidinosuccinate-evoked current through the action of spermine on the polyamine binding site of the NMDA receptor complex, whereas current evoked by high (mmol/L) concentrations of spermine alone involved direct activation of voltage-gated Ca2+ channels. Finally, intracerebroventricular administration of 0.25 micromol/L spermine potentiated clonic convulsions induced by guanidinosuccinate. These neuroexcitatory and synergistic effects of guanidinosuccinate and spermine could take place at pathophysiologic concentrations. CONCLUSION: The observed in vitro and in vivo effects of uremic retention solutes suggest that the identified compounds could play a significant role in uremic pathophysiology. Some of the compounds tested displayed in vitro and in vivo neuroexcitatory effects that were mediated by ligand- and voltage-gated Ca2+ channels. The findings suggest a mechanism for the involvement of calcium toxicity in the central nervous system complications in renal failure with particular reference to guanidinosuccinate and spermine.