Renal cell carcinoma risk in type 2 von Hippel-Lindau disease correlates with defects in pVHL stability and HIF-1alpha interactions.

The von Hippel-Lindau (VHL) tumor suppressor protein is the substrate binding subunit of the CBC(VHL) E3 ubiquitin ligase complex. Mutations in the VHL gene cause a variety of tumors with complex genotype/phenotype correlations. Type 2A and type 2B VHL disease are characterized by a low or high risk of renal cell carcinoma, respectively. To investigate the molecular basis underlying the difference between disease types 2A and 2B, we performed a detailed biochemical analysis of the two most frequent type 2A mutations, Y98 H and Y112 H, in comparison to type 2B mutations in the same residues, Y98N and Y112N. While none of these mutations affected the assembly of CBC(VHL) complexes, the type 2A mutant proteins exhibited higher stabilities at physiological temperature. Moreover, the type 2A mutant proteins possessed higher binding affinities for the key cellular substrate, hypoxia-inducible transcription factor 1 (HIF-1alpha). Consistent with these results, type 2A but not type 2B mutant VHL proteins retained significant ubiquitin ligase activity towards HIF-1alpha in vitro. We propose that this residual ubiquitin ligase activity is sufficient to suppress renal cell carcinogenesis in vivo.

Inhibition of dimethylnitrosamine metabolism by some heterocyclic compounds and by substrates and inhibitors of monoamine oxidase in the rat.

Pretreatment of rats with a number of nitrogen-containing heterocyclic compounds was found to inhibit markedly the metabolism of dimethylnitrosamine (DMN) in terms of both CO2 excretion and decline in blood DMN concentration. However, many of these compounds had either much less or no inhibitory effect on the in vivo metabolism to CO2 of a typical mixed-function oxidase substrate, aminopyrine. In addition, a number of model inhibitors of monoamine oxidase (MAO) activity also inhibited DMN metabolism in the intact animal, and a number of primary amines, known substrates of hepatic MAO, inhibited DMN metabolism but not that of aminopyrine in the isolated perfused liver system. These results, together with in vitro data and previously reported studies on the effect of MAO inhibitors and substrates on the mutagenicity of DMN, suggest that the metabolism and bioactivation of DMN may be in part mediated by a MAO type of enzyme activity.

[Relationship between pre-dialysis period and mortality and morbidity; the importance of timely pre-dialysis care]. / Relatie predialyseduur met mortaliteit en morbiditeit; het belang van een tijdige voorbereiding op dialyse.

OBJECTIVE: To investigate if the duration of pre-dialysis nephrology care is a predictive factor for mortality and morbidity in the first year of renal replacement therapy (RRT). DESIGN: Cohort study. METHOD: We included all patients with chronic or acute-on-chronic renal failure whose estimated glomerular filtration time (eGFR) was < 30 ml/min/1.73 m2 6 months before starting RRT and in whom RRT was initiated in 2005-2006 or 2009-2010. Depending on the duration of the pre-dialysis period we allocated patients to the short (< 6 months) or the long (≥ 6 months) pre-dialysis group. Data regarding mortality and morbidity were registered at the initiation of RRT (T0), after 3 (T3), 6 (T6) and 12 (T12) months. RESULTS: Thirty-nine patients with a short pre-dialysis period and 49 patients with a long pre-dialysis period were included. Patients with a short pre-dialysis period had higher mortality (T6: 23.1% vs. 8.2%; p = 0.05), more hospital stays (2 vs. 1 stay; p = 0.02), and longer hospital stays (16 vs. 3 days; p < 0.01). Additionally, in this group RRT more often had to be started through an acute route of administration for dialysis, which was associated with a higher mortality at T6 (23.8% vs. 6.5%; p = 0.02). CONCLUSION: A too short pre-dialysis period is predictive for higher mortality and morbidity in the first year after initiation of RRT. The necessity for an acute route of administration for dialysis seems to be the most important predictor.

Studies on the mechanism of diet-induced nephrocalcinosis: calcium and phosphorus metabolism in the female rat.

The disposition of calcium and phosphorus in female Sprague-Dawley rats fed either a low- or a high-calcium semi-synthetic diet for up to 9 wk from weaning has been investigated. The rats fed the low-calcium diet (calcium to phosphorus ratio approximately 1:1) developed cortico-medullary nephrocalcinosis within 6 wk, whereas those fed the high-calcium diet (calcium to phosphorus ratio greater than 1.5:1) did not develop any lesion during this time. Intake and excretion of calcium was greater at all times in the animals fed the high-calcium diet than in those on the low-calcium diet. However, the true absorption and retention of calcium was not significantly different between the two diet groups. Phosphorus intake and total excretion were similar for both groups of animals, although urinary excretion accounted for less than 1% of the total in the animals fed the high-calcium diet but more than 50% in those fed the low-calcium diet. Bioavailability of phosphorus decreased with age in both groups. Recovery of injected 45Ca in urine and faeces increased during the experimental period from approximately 3 to 11% of the dose in rats on the low-calcium diet and from approximately 9 to 13% in the high-calcium group. Recovery of both injected and orally administered 32P was substantially greater in the low-calcium group than in the high-calcium group at wk 3 and 5, but at wk 9 was only greater after intramuscular administration. At the earlier times the urine was the major route of 32P excretion. It appears that no gross disturbances of calcium or phosphorus metabolism occurred in female rats maintained on a diet that resulted in the rapid development of kidney calcification.

Metabolic disposition of 14C-labelled carmoisine in the rat, mouse and guinea-pig.

The absorption, metabolism and excretion of 14C-labelled carmoisine has been studied in the rat, mouse and guinea-pig. Following administration of a single oral dose of either 0.5 or 50 mg/kg body weight, substantially all of the dose was recovered in the excreta within 72 hr, mainly in the faeces. Although the urinary excretion of radioactivity was similar in the rat and the mouse, the proportion of the radioactivity found in the urine of the guinea-pig was significantly greater than that of the other species at both dose levels. Pretreating male rats with unlabelled colouring in the diet (0.05%, w/w) for 28 days prior to dosing with 14C-labelled colouring had no effect on the route of excretion or the time taken to eliminate the majority of the labelled dose. Following a single oral dose of 14C-labelled colouring to previously untreated rats, mice and guinea-pigs or to rats pretreated as above, no marked accumulation of radioactivity in any tissue was found. Pregnant rats eliminated a single oral dose of 14C-labelled colouring at a similar rate to non-pregnant females, and the concentration of radioactivity in the foetuses was similar to that in the other tissues. Naphthionic acid was the major urinary metabolite in all three species. In the rat and mouse, most of the remaining radioactivity co-chromatographed with 2-amino-1-naphthol-4-sulphonic acid (2-ANS), but in the guinea-pig radioactivity also co-chromatographed with 1,2-naphthoquinone-4-sulphonate (1,2-NQS). Only a trace amount of unchanged carmoisine was detected in the urine of the species examined. Naphthionic acid was also found in the faeces of all three species, but neither carmoisine, 2-ANS or 1,2-NQS was detected. At least five other radioactive metabolites were found in the faecal extracts of all three species, including a substantial amount of a compound with chromatographic properties similar to those of a trace metabolite in the urine. Two of the faecal metabolites were hydrolysed by beta-glucuronidase and sulphatase treatment. In studies on the absorption of carmoisine at concentrations of 50, 500 or 5000 ppm from isolated intestinal loops, no significant absorption was detected in the rat, mouse or guinea-pig.

The metabolic disposition of 14C-labelled Ponceau 4R in the rat, mouse and guinea-pig.

The absorption, metabolism and excretion of 14C-labelled Ponceau 4R has been studied in the rat, mouse and guinea-pig. Following administration of a single oral dose of 0.5 or 50 mg/kg body weight substantially all of the dose was excreted in the urine and faeces within 72 hr, with the majority being accounted for in the faeces. In all three species, naphthionic acid was the major urinary metabolite, whereas in the faeces naphthionic acid, 7-hydroxy-8-aminonaphthalene-1,3-disulphonic acid and unchanged dye were found. Pretreating male rats with unlabelled Ponceau 4R in the diet (50 mg/kg/day) for 28 days prior to dosing with the 14C-labelled colouring had no effect on the route of excretion or the time taken to eliminate the majority of the label. Following a single dose of 14C-labelled colouring to previously untreated rats, mice and guinea-pigs or to rats pretreated as above, no marked accumulation of radioactivity in any tissue was found, although tissue levels of radioactivity at 72 hr after dosing were higher in the pretreated rats than in those that were not pretreated. Pregnant rats eliminated a single oral dose of 14C-labelled colouring at a similar rate to non-pregnant females; however, some retention of radioactivity in the foetuses was found. In studies of absorption from isolated loops of small intestine containing 50, 500 or 5000 ppm Ponceau 4R, no significant absorption was detected in rats, but some absorption was seen in mice at the lowest concentration, and in the guinea-pig at the two higher concentrations.

Metabolic disposition of 14C-labelled amaranth in the rat, mouse and guinea-pig.

The absorption, metabolism and excretion of orally administered 14C-labelled amaranth has been studied in the rat, mouse and guinea-pig. Following administration of a single oral dose of either 2 or 200 mg/kg, most of the radioactivity was excreted in the urine and faeces in the first 24 hr, and substantially all of the dose was recovered in the excreta within 72 hr. In the rat and mouse, the principal route of excretion was the faeces, whereas in the guinea-pig, urinary excretion accounted for up to 50% of the dose. In the rat and guinea-pig the proportion of the dose excreted in the urine was significantly greater at the lower dose level. No marked accumulation of radioactivity was found in any tissues 72 hr after the administration of the labelled colouring. For all three species most of the radioactivity was shown to be associated with naphthionic acid, with traces of unchanged amaranth and a number of other unidentified metabolites also being detected. In the rat and mouse substantially all of the remaining radioactivity was associated with a single unidentified component. Naphthionic acid was found in the faeces of all three species along with a substantial, but variable, amount of unchanged dye. At least six other radioactive peaks were seen in the chromatograms of faecal extracts; two of these peaks had similar chromatographic properties to the unknown metabolites in the urine, but there was no peak corresponding to 1-amino-2-naphthol-3,6-disulphonic acid (1-ANDSA), previously reported as a urinary metabolite of amaranth. In studies of absorption from isolated loops of small intestine of the rat, mouse and guinea-pig, no significant absorption of amaranth was detected over a 100-fold concentration range (20-2000 ppm).