Bruce Barshop

Metabolomics Reveals Signature of Mitochondrial Dysfunction in Diabetic Kidney Disease

Diabetic kidney disease is the leading cause of ESRD, but few biomarkers of diabetic kidney disease are available. This study used gas chromatography-mass spectrometry to quantify 94 urine metabolites in screening and validation cohorts of patients with diabetes mellitus (DM) and CKD(DM+CKD), in patients with DM without CKD (DM-CKD), and in healthy controls. Compared with levels in healthy controls, 13 metabolites were significantly reduced in the DM+CKD cohorts (P≤0.001), and 12 of the 13 remained significant when compared with the DM-CKD cohort. Many of the differentially expressed metabolites were water-soluble organic anions. Notably, organic anion transporter-1 (OAT1) knockout mice expressed a similar pattern of reduced levels of urinary organic acids, and human kidney tissue from patients with diabetic nephropathy demonstrated lower gene expression of OAT1 and OAT3. Analysis of bioinformatics data indicated that 12 of the 13 differentially expressed metabolites are linked to mitochondrial metabolism and suggested global suppression of mitochondrial activity in diabetic kidney disease. Supporting this analysis, human diabetic kidney sections expressed less mitochondrial protein, urine exosomes from patients with diabetes and CKD had less mitochondrial DNA, and kidney tissues from patients with diabetic kidney disease had lower gene expression of PGC1α (a master regulator of mitochondrial biogenesis). We conclude that urine metabolomics is a reliable source for biomarkers of diabetic complications, and our data suggest that renal organic ion transport and mitochondrial function are dysregulated in diabetic kidney disease.

Decreased Renal Organic Anion Secretion and Plasma Accumulation of Endogenous Organic Anions in OAT1 Knock-out Mice

The “classical” organic anion secretory pathway of the renal proximal tubule is critical for the renal excretion of the prototypic organic anion, para-aminohippurate, as well as of a large number of commonly prescribed drugs among other significant substrates. Organic anion transporter 1 (OAT1), originally identified as NKT (Lopez-Nieto, C. E., You, G., Bush, K. T., Barros, E. J. G., Beier, D. R., and Nigam, S. K. (1997) J. Biol. Chem. 272, 6471–6478), has physiological properties consistent with a role in this pathway. However, several other transporters (e.g. OAT2, OAT3, and MRP1) have also been proposed as important PAH transporters on the basis of in vitro studies; therefore, the relative contribution of OAT1 has remained unclear. We have now generated a colony of OAT1 knock-out mice, permitting elucidation of the role of OAT1 in the context of these other potentially functionally redundant transporters. We find that the knock-out mice manifest a profound loss of organic anion transport (e.g. para-aminohippurate) both ex vivo (in isolated renal slices) as well as in vivo (as indicated by loss of renal secretion). In the case of the organic anion, furosemide, loss of renal secretion in the knock-out results in impaired diuretic responsiveness to this drug. These results indicate a critical role for OAT1 in the functioning of the classical pathway. In addition, we have determined the levels of ∼60 endogenous organic anions in the plasma and urine of wild-type and knock-out mice. This has led to identification of several compounds with significantly higher plasma concentrations and/or lower urinary concentrations in knock-out mice, suggesting the involvement of OAT1 in their renal secretion. We have also demonstrated in xenopus oocytes that some of these compounds interact with OAT1 in vitro. Thus, these latter compounds might represent physiological substrates of OAT1.

Autism Associated With the Mitochondrial DNA G8363A Transfer RNALys Mutation

We report a family with a heterogeneous group of neurologic disorders associated with the mitochondrial DNA G8363A transfer ribonucleic acid (RNA)Lys mutation. The phenotype of one child in the family was consistent with autism. During his second year of life, he lost previously acquired language skills and developed marked hyperactivity with toe-walking, abnormal reciprocal social interaction, stereotyped mannerisms, restricted interests, self-injurious behavior, and seizures. Brain magnetic resonance imaging (MRI) and repeated serum lactate studies were normal. His older sister developed signs of Leigh syndrome with progressive ataxia, myoclonus, seizures, and cognitive regression. Her laboratory studies revealed increased MRI T2-weighted signal in the putamen and posterior medulla, elevated lactate in serum and cerebrospinal fluid, and absence of cytochrome c oxidase staining in muscle histochemistry. Molecular analysis in her revealed the G8363A mutation of the mitochondrial transfer RNA Lys gene in blood (82% mutant mitochondrial DNA) and muscle (86%). The proportions of mutant mitochondrial DNA from her brother with autism were lower (blood 60%, muscle 61%). It is likely that the origin of his autism phenotype is the pathogenic G8363A mitochondrial DNA mutation. This observation suggests that certain mitochondrial point mutations could be the basis for autism in some individuals. (J Child Neurol 2000;15:357-361).

Agalsidase Alfa and Kidney Dysfunction in Fabry Disease

In male patients with Fabry disease, an X-linked disorder of glycosphingolipid metabolism caused by deficient activity of the lysosomal enzyme alpha-galactosidase A, kidney dysfunction becomes apparent by the third decade of life and invariably progresses to ESRD without treatment. Here, we summarize the effects of agalsidase alfa on kidney function from three prospective, randomized, placebo-controlled trials and their open-label extension studies involving 108 adult male patients. The mean baseline GFR among 54 nonhyperfiltrating patients (measured GFR <135 ml/min per 1.73 m(2)) treated with placebo was 85.4 +/- 29.6 ml/min per 1.73 m(2); during 6 mo of placebo, the mean annualized rate of change in GFR was -7.0 +/- 32.9 ml/min per 1.73 m(2). Among 85 nonhyperfiltrating patients treated with agalsidase alfa, the annualized rate of change was -2.9 +/- 8.7 ml/min per 1.73 m(2). Treatment with agalsidase alfa did not affect proteinuria. Multivariate analysis revealed that GFR and proteinuria category (< 1 or > or = 1 g/d) at baseline significantly predicted the rate of decline of GFR during treatment. This summary represents the largest group of male patients who had Fabry disease and for whom the effects of enzyme replacement therapy on kidney function have been studied. These data suggest that agalsidase alfa may stabilize kidney function in these patients.

Effects of Ascorbic Acid in Alkaptonuria: Alterations in Benzoquinone Acetic Acid and an Ontogenic Effect in Infancy

ABSTRACT: The effects of ascorbic acid on the excretion of homogentisic acid and its derivative benzoquinone acetic acid were studied in two adults and three infants. The administration of relatively large amounts of ascorbic acid to the adults was followed by a disappearance of benzoquinone acetic acid from the urine, whereas the level of excretion of homogentisic acid did not change. This could have relevance to the pathogenesis of ochronotic arthritis. In the 4-mo-old infant and the 5-mo-old infant ascorbic acid in the urine may have doubled the amount of homogentisic acid, presumably through an effect on the immature p-hydroxyphenylpyruvic acid oxidase. Dietary reduction of the intake of tyrosine and phenylalanine substantially reduced the excretion of homogentisic acid.

Exome sequencing identifies ACSF3 as a cause of combined malonic and methylmalonic aciduria

We used exome sequencing to identify the genetic basis of combined malonic and methylmalonic aciduria (CMAMMA). We sequenced the exome of an individual with CMAMMA and followed up with sequencing of eight additional affected individuals (cases). This included one individual who was identified and diagnosed by searching an exome database. We identify mutations in ACSF3, encoding a putative methylmalonyl-CoA and malonyl-CoA synthetase as a cause of CMAMMA. We also examined a canine model of CMAMMA, which showed pathogenic mutations in a predicted ACSF3 ortholog. ACSF3 mutant alleles occur with a minor allele frequency of 0.0058 in ∼1,000 control individuals, predicting a CMAMMA population incidence of ∼1:30,000. ACSF3 deficiency is the first human disorder identified as caused by mutations in a gene encoding a member of the acyl-CoA synthetase family, a diverse group of evolutionarily conserved proteins, and may emerge as one of the more common human metabolic disorders.

Organic Anion Transporter 3 Contributes to the Regulation of Blood Pressure

Renal organic anion transporters (OAT) are known to mediate the excretion of many drugs, but their function in normal physiology is not well understood. In this study, mice lacking organic anion transporter 3 (Oat3) had a 10 to 15% lower BP than wild-type mice, raising the possibility that Oat3 transports an endogenous regulator of BP. The aldosterone response to a low-salt diet was blunted in Oat3-null mice, but baseline aldosterone concentration was higher in these mice, suggesting that aldosterone dysregulation does not fully explain the lower BP in the basal state; therefore, both targeted and global metabolomic analyses of plasma and urine were performed, and several potential endogenous substrates of Oat3 were found to accumulate in the plasma of Oat3-null mice. One of these substrates, thymidine, was transported by Oat3 expressed in vitro. In vivo, thymidine, as well as two of the most potent Oat3 inhibitors that were characterized, reduced BP by 10 to 15%; therefore, Oat3 seems to regulate BP, and Oat3 inhibitors might be therapeutically useful antihypertensive agents. Moreover, polymorphisms in human OAT3 might contribute to the genetic variation in susceptibility to hypertension.

Luminescent immobilized enzyme test systems for inorganic pyrophosphate: Assays using firefly luciferase and nicotinamide-mononucleotide adenylyl transferase or adenosine-5′-triphosphate sulfurylase

Inorganic pyrophosphate was measured by luminescence produced by a pyrophosphatase (NAD adenylyl-transferase or ATP sulfurylase) coimmobilized with firefly luciferase on Sepharose beads, with continuous flow of saturating concentrations of substrates (NAD plus luciferin or adenylophosphosulfate plus luciferin, respectively) and intermittent injections of samples containing pyrophosphate. In this scheme, the limiting substrate (pyrophosphate) is regenerated, a situation that is well suited to a bioluminescent assay. The instrumentation allowed for automation with a through-put of approximately one sample every 4 min. With standard solutions or samples that do not contain ATP, the sensitivity of the assay permits detection of less than 1 pmol pyrophosphate in a volume of 20 microliters (50 nmol/liter) with a coefficient of variation approximately equal to 4%. To assay biological samples, it was shown that endogenous ATP can be inactivated by oxidation with sodium periodate. Periodate treatment and quenching engenders dilution that limits the sensitivity to approximately 600 nmol/liter pyrophosphate in the starting material. The assay has been applied to the determination of intracellular pyrophosphate in human lymphocytes and to the measurement of nucleoside-triphosphate pyrophosphohydrolase in human fibroblasts. The variability of the assay was greater with biological samples than with standard samples, with a coefficient of variation of 15.3% in a series of determinations of intracellular pyrophosphate in a series of replicate lymphocyte lysates. Bioluminescent systems of coupled coimmobilized enzymes offer great promise for sensitive, safe, automated assaying of metabolites.

Ascorbate decreases fabry cerebral hyperperfusion suggesting a reactive oxygen species abnormality: An arterial spin tagging study

To test the hypothesis that reactive oxygen species contribute to the cerebral hyperperfusion in Fabry disease.

Domino liver transplantation in maple syrup urine disease

Liver transplantation has been reported in a few cases of maple syrup urine disease (MSUD), but is controversial. Many patients with approved indications for liver transplantation die before grafts are available. A 25‐yr‐old man with MSUD underwent liver transplantation, and his liver was used as a domino graft for a 53‐yr‐old man with hepatocellular carcinoma who had low priority on the liver transplant waiting list and was unlikely to survive until routine organ procurement. Both transplants were performed as “piggy back” procedures, reconstructing the domino graft with caval segments from the cadaveric donor. Neither required veno‐venous bypass. Whole body leucine oxidation was estimated by 13CO2 in breath after oral boluses of L‐[1‐13C]‐leucine, before and after transplantation in both patients and a control subject. The surgical outcome was successful. The patient with MSUD had marked decreases in plasma branched‐chain amino acids (BCAAs) and alloisoleucine (from 255 ± 66 to 16 ± 7 μmol/L), despite advancement of dietary protein from 6 to >40 gm/day. The domino recipient maintained near‐normal levels of plasma amino acids with no detectable alloisoleucine on unrestricted diet. Leucine oxidation increased in the patient with MSUD (from 2.2 to 5.6% recovered in 4 hours) and decreased in the recipient (from 9.7 to 6.2%). Neither patient demonstrated any apparent symptoms of MSUD over more than 7 months. In conclusion, liver transplantation substantially corrects whole body BCAA metabolism in MSUD and greatly attenuates the disease. Livers from patients with MSUD may be considered as domino grafts for patients who might otherwise not survive until transplantation. Liver Transpl 12:876–882, 2006.