Oliver Wrong

UTILISATION OF AMMONIA NITROGEN FOR PROTEIN SYNTHESIS IN MAN, AND THE EFFECT OF PROTEIN RESTRICTION AND URÆMIA

Abstract The effect of dietary protein restriction on the incorporation of 15 N-ammonia into body protein has been studied in 8 experiments on four healthy individuals and two uraemic patients. In healthy individuals, the more severe the protein restriction the greater was the incorporation of the isotope into the plasma-albumin pool, and the smaller the recovery of 15 N in urine and faeces. The two stable uraemic patients who had been protein restricted for 9 months incorporated between three and five times as much ammonia nitrogen into the plasma-albumin as did two healthy individuals after 3 weeks on a 20 g. protein intake, with a total calculated incorporation of 700 and 975 mg. of ammonia nitrogen in 24 hours, equivalent to about 4.5 and 6.0 g. of protein, respectively. Evidence is cited that ammonia is the form in which man reincorporates urea nitrogen into protein, and that this ammonia is derived from urea by bacterial breakdown in the intestine. We suggest that the ability of uraemic patients to re-utilise their urea nitrogen in protein synthesis could be exploited by providing them with an artificial diet containing the ketoacid analogues of the essential aminoacids, but no other nitrogen except for lysine and threonine, which may not be formed in sufficient amount in the body from their ketoacid analogues.

OCRL1 Mutations in Dent 2 Patients Suggest a Mechanism for Phenotypic Variability

Background/Aims: Dent disease is an X-linked renal proximal tubulopathy associated with mutations in CLCN5 (Dent 1) or OCRL1 (Dent 2). OCRL1 mutations also cause the oculocerebrorenal syndrome of Lowe. Methods: Dent patients with normal sequence for CLCN5 were sequenced for mutations in OCRL1. By analyzing these and all other OCRL1 mutations reported, a model relating OCRL1 mutations to the resulting disease (Dent 2 or Lowe’s) was developed. Results: Six boys with Dent disease had novel OCRL1 mutations: two missense (R301H, G304E) and four mutations predicted to produce premature termination codons (L56DfsX1, S149X, P161PfsX3, and M170IfsX1). These include one of the original patients reported by Dent and Friedman. Slit lamp examinations revealed early cataracts in only one boy with normal vision. None of these Dent 2 patients had metabolic acidosis; 3 had mild mental retardation. Analysis of all known OCRL1 mutations show that Dent 2 mutations fall into two classes that do not overlap with Lowe mutations. Bioinformatics analyses identified expressed OCRL1 splice variants that help explain the variability of those clinical features that distinguish Dent disease from Lowe syndrome. Conclusions:OCRL1 mutations can cause the renal phenotype of Dent disease, without acidosis or the dramatic eye abnormalities typical of Lowe syndrome. We propose a model to explain the phenotypic variability between Dent 2 and Lowe’s based on distinctly different classes of mutations in OCRL1 producing splice variants.

Receptor-mediated endocytosis and endosomal acidification is impaired in proximal tubule epithelial cells of Dent disease patients

Receptor-mediated endocytosis, involving megalin and cubilin, mediates renal proximal-tubular reabsorption and is decreased in Dent disease because of mutations of the chloride/proton antiporter, chloride channel-5 (CLC-5), resulting in low-molecular-weight proteinuria, hypercalciuria, nephrolithiasis, and renal failure. To facilitate studies of receptor-mediated endocytosis and the role of CLC-5, we established conditionally immortalized proximal-tubular epithelial cell lines (ciPTECs) from three patients with CLC-5 mutations (30:insH, R637X, and del132-241) and a normal male. Confocal microscopy using the tight junction marker zona occludens-1 (ZO-1) and end-binding protein-1 (EB-1), which is specific for the plus end of microtubules demonstrated that the ciPTECs polarized. Receptor-mediated endocytic uptake of fluorescent albumin and transferrin in 30:insH and R637X ciPTECs was significantly decreased, compared with normal ciPTECs, and could be further reduced by competition with 10-fold excess of unlabeled albumin and transferrin, whereas in the del132-241 ciPTEC, receptor-mediated endocytic uptake was abolished. Investigation of endosomal acidification by live-cell imaging of pHluorin-VAMP2 (vesicle-associated membrane protein-2), a pH-sensitive-GFP construct, revealed that the endosomal pH in normal and 30:insH ciPTECs was similar, whereas in del132-241 and R637X ciPTECs, it was significantly more alkaline, indicating defective acidification in these ciPTECs. The addition of bafilomycin-A1, a V-ATPase inhibitor, raised the pH significantly in all ciPTECs, demonstrating that the differences in acidification were not due to alterations in the V-ATPase, but instead to abnormalities of CLC-5. Thus, our studies, which have established human Dent disease ciPTECs that will facilitate studies of mechanisms in renal reabsorption, demonstrate that Dent disease-causing CLC-5 mutations have differing effects on endosomal acidification and receptor-mediated endocytosis that may not be coupled.

Urea and ammonia metabolism in the human large intestine

Man passes each day 100-200 g faeces containing 1-2 g bound nitrogen. The chemical nature of this N is not completely clear, although recent studies suggest that it lies predominantly in faecal bacteria, which collectively make up 40-7070 of faecal wet weight (Stephen & Cummings, 1980). The work we shall present here is concerned with the analysis of this faecal N, and here one immediately comes up against a technical problem: how does one analyse labile nitrogenous substances such as urea and ammonia in an offensive, variably-coloured material which has the consistency of a solid despite being 75% water, and is, moreover, the site of intense bacterial activity? Over 20 years ago the technique of in vivo faecal dialysis (Wrong et al. 1961, 1965) was developed to deal with this problem. The experimental subject swallows small dialysis capsules made of cellulose tubing, and 24-120 h later these are recovered when passed in the stool. Except in diarrhoeal states the capsules spend at least 20 h in the large intestine which is ample time to achieve diffusion equilibrium with large bowel contents. The fluid contained in the capsules is a dialysate of distal faeces and can be analysed by conventional techniques. The method samples only the extracellular component of stool water and not the water enclosed in the cell membranes of bacteria and epithelial cells. Other workers have obtained faecal water by fractionation of faeces outside the body, using high-speed centrifugation (Tarlow & Thom, 1974) or ultrafiltration (Bjork et al. 1976). These methods produce fluids of similar composition to in vivo dialysate with respect to inorganic electrolytes, but of higher ammonia and organic acid concentrations and lower pH and bicarbonate concentration (Table I). Our own studies, some of which we describe here, show that these differences arise

Renal Chloride Channel, CLCN5, Mutations in Dent's Disease

Dents disease is an X‐linked renal tubular disorder characterized by low‐molecular‐weight proteinuria, hypercalciuria, nephrocalcinosis, nephrolithiasis, and renal failure. Patients with Dents disease may also suffer from rickets and other features of the renal Fanconi Syndrome. Patients may have mutations in the X‐linked renal chloride channel gene, CLCN5, which encodes a 746‐amino‐acid protein with 12–13 transmembrane domains. We have investigated the 11 coding exons of CLCN5 for mutations in eight unrelated patients with Dents disease. Leukocyte DNA was used for the polymerase chain reaction amplification of CLCN5 and the products analyzed for single‐stranded conformational polymorphisms (SSCPs). Abnormal SSCPs were sequenced and revealed eight mutations. These consisted of three nonsense mutations (Arg34Stop, Arg648Stop, Arg704Stop), four deletions involving codons 40, 86, 157, and 241, and one acceptor splice consensus sequence mutation tgcag → tgaag. The mutations were confirmed either by restriction endonuclease or sequence‐specific oligonucleotide hybridization analysis. In addition, an analysis of 110 alleles from 74 unrelated normal individuals demonstrated that the DNA sequence changes were not common polymorphisms. All of the mutations predict truncated chloride channels that are likely to result in a functional loss. Thus, our findings expand the spectrum of CLCN5 mutations associated with Dents disease and the results will help to elucidate further the functional domains of this novel chloride channel.

Recessive distal renal tubular acidosis in Sarawak caused by AE1 mutations

Mutations of the AE1 (SLC4A1, Anion-Exchanger 1) gene that codes for band 3, the renal and red cell anion exchanger, are responsible for many cases of familial distal renal tubular acidosis (dRTA). In Southeast Asia this disease is usually recessive, caused either by homozygosity of a single AE1 mutation or by compound heterozygosity of two different AE1 mutations. We describe two unrelated boys in Sarawak with dRTA associated with compound heterozygosity of AE1 mutations. Both had Southeast Asian ovalocytosis (SAO), a morphological abnormality of red cells caused by a deletion of band 3 residues 400–408. In addition, one boy had a DNA sequence abnormality of band 3 residue (G701D), which has been reported from elsewhere in Southeast Asia. The other boy had the novel sequence abnormality of band 3 (Q759H) and profound hemolytic anemia.

Sevelamer and Other Anion-Exchange Resins in the Prevention and Treatment of Hyperphosphataemia in Chronic Renal Failure

Sevelamer, or more precisely ‘sevelamer hydrochloride’, is a weakly basic anion-exchange resin in the chloride form that was introduced in 1997 for the treatment of the hyperphosphataemia of patients with end-stage renal failure, usually those on long-term haemodialysis. The rationale for this therapy was that sevelamer would sequester phosphate within the gastrointestinal tract, so preventing its absorption and enhancing its faecal excretion. Over the succeeding years, large numbers of patients have been treated with sevelamer, and it has fulfilled expectations in helping to control the hyperphosphataemia of end-stage renal failure. However, it is only one of many anion-exchange resins that could be used for this purpose, some of which are currently available for clinical use and are much less costly than sevelamer. Theoretical considerations suggest that some of these other resins might be at least as efficient as sevelamer in sequestering phosphate in the gastrointestinal tract. Neither sevelamer, nor any of these other agents, has been submitted to a proper metabolic balance study to measure the amount of phosphate sequestered by the resin in the bowel, and without this information it is impossible to judge which is the ideal resin for this purpose.

DOMESTIC METALLIC MERCURY POISONING

In a family exposed to metallic mercury vapour two patients had acrodynia, one had the nephrotic syndrome, and one person remained well. Recognition of the variable manifestations of the disease and prevention of further exposure were the most important aspects of management. Recovery appeared to be complete as blood mercury levels fell to normal. Urinary mercury levels were too variable to be reliable as indications of progress.

The association between familial distal renal tubular acidosis and mutations in the red cell anion exchanger (band 3, AE1) gene.

In distal renal tubular acidosis (dRTA) the tubular secretion of hydrogen ion in the distal nephron is impaired, leading to the development of metabolic acidosis, frequently accompanied by hypokalemia, nephrocalcinosis, and metabolic bone disease. The condition can be familial, when it is usually inherited as an autosomal dominant, though there is a rarer autosomal recessive form associated with nerve deafness. It has been shown that the autosomal dominant form of dRTA is associated with a defect in the anion exchanger (AE1) of the renal collecting duct intercalated cell. This transporter is a product of the same gene (AE1) as the erythrocyte anion exchanger, band 3. In this review we will look at the evidence for this association. Studies of genomic DNA from families with this disorder have shown, both by genetic linkage studies and by DNA sequencing, that affected individuals are heterozygous for mutations in the AE1 gene whilst unaffected family members have a normal band 3 sequence. Mutations have been found in the region of proposed helices 6 and 7 of the membrane domain of band 3 and involve amino acids Arg-589 and Ser-613, and in the COOH-terminal domain of band 3. Studies of red cell band 3 from these families have provided information on the effect these mutations have on the structure and function of erythrocyte band 3. Expression studies of the erythroid and kidney isoforms of the mutant AE1 proteins, in Xenopus laevis oocytes, have shown that they retained chloride transport activity, suggesting that the disease in the dRTA families is not related simply to the anion transport activity of the mutated proteins. A possible explanation for the dominant effect of these mutant AE1 proteins in the kidney cell is that these mutations affect the targeting of AE1 from the basolateral to the apical membrane of the alpha-intercalated cell.

Distal renal tubular acidosis: the value of urinary pH,PCO2 and NH4+ measurements

Distal renal tubular acidosis (dRTA) is not a single disease. The experimental forms of the syndrome are unsatisfactory as models of the naturally occurring disease, not least because they are seldom complicated by nephrocalcinosis, which is present in the majority of patients with spontaneous disease and contributes to the renal tubular defects found in the syndrome. Impairment of minimal urine pH, reduced urine carbon dioxide tension (PCO2) during passage of alkaline urine, and reduced urinary ammonium (NH4+) excretion, have all been advocated as essential criteria for the diagnosis of dRTA. Minimal urine pH, measured during metabolic acidosis, sulphate infusion, or after oral frusemide, is the yardstick against which other criteria should be assessed. A reduced urinaryPCO2 is commonly found in dRTA but is not specific for the syndrome and may be accounted for by tubular defects other than those involving reduced distal hydrogen ion secretion. NH4+ excretion is reduced in most patients with renal acidosis whatever the nature of the underlying renal disease; this function is closely related to nephron mass, and is not specifically impaired in renal tubular disease.