Jon A. Gangoiti

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.

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.

Twice-Daily Cysteamine Bitartrate Therapy for Children with Cystinosis

OBJECTIVE Cystinosis causes renal and other organ failure. Regular 6-hourly cysteamine bitartrate (Cystagon; Mylan, Morgantown, West Virginia) reduces intracellular cystine and the rate of organ deterioration. A formulation of cysteamine requiring less frequent dosing may improve compliance and possibly patient outcome. METHODS Enteric-release cysteamine was prepared. For a period of 1 month, patients received their regular cysteamine dose every 6 hours (stage I). The patients then underwent pharmacokinetic and pharmacodynamic studies following washout periods using single-doses of cysteamine and enteric-release cysteamine (stage II). Finally, the patients commenced regular enteric-release cysteamine therapy (stage III). Weekly trough white blood cell (WBC) cystine levels were recorded. RESULTS Seven children with cystinosis (mean age, 11.8 years; range, 8-17 years) who received cysteamine and enteric-release cysteamine (mean dose, 45 and 28.8 mg/kg body weight/day, respectively) had mean WBC cystine levels of 0.7+/-0.3 and 0.41+/-0.22 nmol half-cystine/mg protein in study stages I and III, respectively. Study stage II showed that the mean time (T(max)) to reach the maximum plasma cysteamine level (C(max)) was longer for enteric-release cysteamine than for cysteamine (176 minutes vs 60 minutes; P=.001), but the mean C(max) at the same dose was similar. Mean serum gastrin levels were similar after ingestion of cysteamine and enteric-release cysteamine. CONCLUSIONS Twelve-hour enteric-release cysteamine, given at approximately 60% of the previous daily dose of cysteamine, was effective in maintaining trough WBC cystine levels within a satisfactory range.

A model‐driven quantitative metabolomics analysis of aerobic and anaerobic metabolism in E. coli K‐12 MG1655 that is biochemically and thermodynamically consistent

The advent of model‐enabled workflows in systems biology allows for the integration of experimental data types with genome‐scale models to discover new features of biology. This work demonstrates such a workflow, aimed at establishing a metabolomics platform applied to study the differences in metabolomes between anaerobic and aerobic growth of Escherichia coli. Constraint‐based modeling was utilized to deduce a target list of compounds for downstream method development. An analytical and experimental methodology was developed and tailored to the compound chemistry and growth conditions of interest. This included the construction of a rapid sampling apparatus for use with anaerobic cultures. The resulting genome‐scale data sets for anaerobic and aerobic growth were validated by comparison to previous small‐scale studies comparing growth of E. coli under the same conditions. The metabolomics data were then integrated with the E. coli genome‐scale metabolic model (GEM) via a sensitivity analysis that utilized reaction thermodynamics to reconcile simulated growth rates and reaction directionalities. This analysis highlighted several optimal network usage inconsistencies, including the incorrect use of the beta‐oxidation pathway for synthesis of fatty acids. This analysis also identified enzyme promiscuity for the pykA gene, that is critical for anaerobic growth, and which has not been previously incorporated into metabolic models of E coli. Biotechnol. Bioeng. 2014;111: 803–815.

Validation of a dual LC–HRMS platform for clinical metabolic diagnosis in serum, bridging quantitative analysis and untargeted metabolomics

Mass spectrometry-based metabolomics is a rapidly growing field in both research and diagnosis. Generally, the methodologies and types of instruments used for clinical and other absolute quantification experiments are different from those used for biomarkers discovery and untargeted analysis, as the former requires optimal sensitivity and dynamic range, while the latter requires high resolution and high mass accuracy. We used a Q-TOF mass spectrometer with two different types of pentafluorophenyl (PFP) stationary phases, employing both positive and negative ionization, to develop and validate a hybrid quantification and discovery platform using LC–HRMS. This dual-PFP LC–MS platform quantifies over 50 clinically relevant metabolites in serum (using both MS and MS/MS acquisitions) while simultaneously collecting high resolution and high mass accuracy full scans to monitor all other co-eluting non-targeted analytes. We demonstrate that the linearity, accuracy, and precision results for the quantification of a number of metabolites, including amino acids, organic acids, acylcarnitines and purines/pyrimidines, meets or exceeds normal bioanalytical standards over their respective physiological ranges. The chromatography resolved highly polar as well as hydrophobic analytes under reverse-phase conditions, enabling analysis of a wide range of chemicals, necessary for untargeted metabolomics experiments. Though previous LC–HRMS methods have demonstrated quantification capabilities for various drug and small molecule compounds, the present study provides an HRMS quant/qual platform tailored to metabolic disease; and covers a multitude of different metabolites including compounds normally quantified by a combination of separate instrumentation.

The effect of different ubiquinones on lifespan in Caenorhabditis elegans

Ubiquinone (UQ, Coenzyme Q, CoQ) transfers electrons from complexes I and II to complex III in the mitochondrial electron transport chain. Depending on the degree of reduction, UQ can act as either a pro- or an antioxidant. Mutations disrupting ubiquinone synthesis increase lifespan in both the nematode (clk-1) and the mouse (mclk-1). The mutated nematodes survive using exogenous ubiquinone from bacteria, which has a shorter isoprenyl tail length (UQ(8)) than the endogenous nematode ubiquinone (UQ(9)). The mechanism underlying clk-1s increased longevity is not clear. Here we directly measure the effect of different exogenous ubiquinones on clk-1 lifespan and mitochondrial function. We fed clk-1 engineered bacteria that produced UQ(6), UQ(7), UQ(8), UQ(9) or UQ(10), and measured clk-1s lifespan, mitochondrial respiration, ROS production, and accumulated ROS damage to mitochondrial protein. Regardless of dietary UQ, clk-1 animals have increased lifespan, decreased mitochondrial respiration, and decreased ROS damage to mitochondrial protein than N2. However, clk-1 mitochondria did not produce less ROS than N2. The simplest explanation of our results is that clk-1 mitochondria scavenge ROS more effectively than wildtype due to the presence of DMQ(9). Moreover, when compared to other dietary quinones, UQ(10) further decreased mitochondrial oxidative damage and extended adult lifespan in clk-1.

Long-Term Treatment of Cystinosis in Children with Twice-Daily Cysteamine

OBJECTIVE Cystinosis causes renal and other organ failure. Treatment with 6-hourly cysteamine bitartrate (Cystagon, Mylan, Morgantown, West Virginia) reduces intracellular cystine and the rate of organ deterioration. A recent study showed that an enteric-release cysteamine required less frequent daily dosing. This report describes the long-term use of enteric-coated (EC) cysteamine bitartrate (Cystagon) in children with cystinosis. STUDY DESIGN After a pharmacokinetic and pharmacodynamic study of EC-cysteamine in children with cystinosis, 5 patients remained on twice-daily treatment. White blood cell cystine levels were measured 12 hours after ingestion every 4 to 8 weeks. These levels were then compared with the patients previous 6-h post-dose levels taken while on regular cysteamine bitartrate before entering the study. Blood chemistry was also measured. RESULTS Five children with cystinosis (mean age, 9 years; range, 8 to 17 years) who previously took cysteamine bitartrate (mean dose, 47 mg/kg body wt), received EC-cysteamine for 10 to 27 months (mean dose, 25 mg/kg body wt) and had mean white blood cell cystine levels of 0.77 and 0.71 nmol half-cystine/mg protein, respectively. During the study period, patients maintained adequate growth and there was no significant deterioration in renal or thyroid function. Two children were required to restart acid suppression after 6 months on EC-cysteamine therapy. CONCLUSIONS Long-term, twice-daily EC-cysteamine, given at approximately 60% of the previous daily dose of cysteamine bitartrate, was effective at maintaining white blood cell cystine levels within a satisfactory range. There was no significant deterioration in renal or thyroid function.

Cysteamine modulates oxidative stress and blocks myofibroblast activity in CKD.

Therapy to slow the relentless expansion of interstitial extracellular matrix that leads to renal functional decline in patients with CKD is currently lacking. Because chronic kidney injury increases tissue oxidative stress, we evaluated the antifibrotic efficacy of cysteamine bitartrate, an antioxidant therapy for patients with nephropathic cystinosis, in a mouse model of unilateral ureteral obstruction. Fresh cysteamine (600 mg/kg) was added to drinking water daily beginning on the day of surgery, and outcomes were assessed on days 7, 14, and 21 after surgery. Plasma cysteamine levels showed diurnal variation, with peak levels similar to those observed in patients with cystinosis. In cysteamine-treated mice, fibrosis severity decreased significantly at 14 and 21 days after unilateral ureteral obstruction, and renal oxidized protein levels decreased at each time point, suggesting reduced oxidative stress. Consistent with these results, treatment of cultured macrophages with cysteamine reduced cellular generation of reactive oxygen species. Furthermore, treatment with cysteamine reduced α-smooth muscle actin-positive interstitial myofibroblast proliferation and mRNA levels of extracellular matrix proteins in mice and attenuated myofibroblast differentiation and proliferation in vitro, but did not augment TGF-β signaling. In a study of renal ischemia reperfusion, cysteamine therapy initiated 10 days after injury and continued for 14 days decreased renal fibrosis by 40%. Taken together, these data suggest previously unrecognized antifibrotic actions of cysteamine via TGF-β-independent mechanisms that include oxidative stress reduction and attenuation of the myofibroblast response to kidney injury and support further investigation into the potential benefit of cysteamine therapy in the treatment of CKD.

Pharmacokinetics of enteric-coated cysteamine bitartrate in healthy adults: a pilot study

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT Cysteamine bitartrate is taken lifelong, every 6 h and for the treatment of cystinosis. Recent studies using cysteamine for for other diseases such as neurodegenerative disorders adopt the same dosing regimen for cysteamine. Regular cysteamine bitartrate (Cystagon) may cause upper gastrointestinal symptoms in some patients. WHAT THIS STUDY ADDS This is the only study that provides pharmacokinetic data for cysteamine delivered in an enteric-release preparation in normal subjects. EC-cysteamine is very well tolerated and does not cause increased gastrin concentrations, even at relatively high doses. EC-cysteamine at the higher dose results in better drug uptake as measured by Cmax and AUC and is more likely to be effective. AIMS Cysteamine bitartrate (Cystagon) is the approved treatment for cystinosis. Poor compliance and patient outcome may occur because the drug needs to be taken every 6 h and in some patients causes gastrointestinal symptoms due to hypergastrinaemia. A formulation of cysteamine requiring twice daily ingestion would improve the quality of life for these patients. This study compares the pharmacokinetics and gastrin production following cysteamine bitartrate non-enteric-coated and cysteamine bitartrate enteric-coated in normal healthy subjects. METHODS Enteric-coated cysteamine was prepared. Following single doses of cysteamine bitartrate non-enteric-coated 450 mg and cysteamine bitartrate enteric-coated 450 mg and 900 mg, serial plasma cysteamine and gastrin concentrations were measured. Two subjects also received cysteamine bitartrate non-enteric-coated 900 mg. Gastrointestinal (GI) symptoms were recorded. RESULTS Six healthy adults (mean age 20.7 years, range 18-24 years; mean weight 59.3 kg) received drug. All post-dose gastrin concentrations were within the normal range (<100 pg ml(-1)). The tmax following cysteamine bitartrate non-enteric-coated (mean and SD is 75+/-19 min) was shorter than cysteamine bitartrate enteric-coated (220+/-74 min) (P=0.001), but only the Cmax and AUC estimates following 900 mg cysteamine bitartrate enteric-coated were significantly greater than any of the other preparations or doses (P<0.05). One patient had GI symptoms following both 900 mg cysteamine bitartrate non-enteric-coated and cysteamine bitartrate enteric-coated. CONCLUSION Although patient numbers were low, single high doses of cysteamine bitartrate enteric-coated were better tolerated than similar doses of cysteamine bitartrate non-enteric-coated in the healthy subjects and all had normal gastrin concentrations. The delayed tmax following cysteamine bitartrate enteric-coated suggested that the cysteamine was released enterically.