Knock-out of dipeptidase CN2 in human proximal tubular cells disrupts dipeptide and amino acid homeostasis and para- and transcellular solute transport.

AIM: Although of potential biomedical relevance, dipeptide metabolism has hardly been studied. We found the dipeptidase carnosinase-2 (CN2) to be abundant in human proximal tubules, which regulate water and solute homeostasis. We therefore hypothesized, that CN2 has a key metabolic role, impacting proximal tubular transport function. METHODS: A knockout of the CN2 gene (CNDP2-KO) was generated in human proximal tubule cells and characterized by metabolomics, RNA-seq analysis, paracellular permeability analysis and ion transport. RESULTS: CNDP2-KO in human proximal tubule cells resulted in the accumulation of cellular dipeptides, reduction of amino acids and imbalance of related metabolic pathways, and of energy supply. RNA-seq analyses indicated altered protein metabolism and ion transport. Detailed functional studies demonstrated lower CNDP2-KO cell viability and proliferation, and altered ion and macromolecule transport via trans- and paracellular pathways. Regulatory and transport protein abundance was disturbed, either as a consequence of the metabolic imbalance or the resulting functional disequilibrium. CONCLUSION: CN2 function has a major impact on intracellular amino acid and dipeptide metabolism and is essential for key metabolic and regulatory functions of proximal tubular cells. These findings deserve in vivo analysis of the relevance of CN2 for nephron function and regulation of body homeostasis.

Slow release of a synthetic auxin induces formation of adventitious roots in recalcitrant woody plants.

Clonal propagation of plants by induction of adventitious roots (ARs) from stem cuttings is a requisite step in breeding programs. A major barrier exists for propagating valuable plants that naturally have low capacity to form ARs. Due to the central role of auxin in organogenesis, indole-3-butyric acid is often used as part of commercial rooting mixtures, yet many recalcitrant plants do not form ARs in response to this treatment. Here we describe the synthesis and screening of a focused library of synthetic auxin conjugates in Eucalyptus grandis cuttings and identify 4-chlorophenoxyacetic acid-L-tryptophan-OMe as a competent enhancer of adventitious rooting in a number of recalcitrant woody plants, including apple and argan. Comprehensive metabolic and functional analyses reveal that this activity is engendered by prolonged auxin signaling due to initial fast uptake and slow release and clearance of the free auxin 4-chlorophenoxyacetic acid. This work highlights the utility of a slow-release strategy for bioactive compounds for more effective plant growth regulation.

Kidney urinary biomarkers in patients with branched-chain amino acid and cobalamin metabolism defects.

There is a clinical need for early detection of chronic kidney disease (CKD) in patients with organic acidurias. We measured kidney markers in a longitudinal study over 5 years in 40 patients with methylmalonic aciduria (Mut0 ), propionic aciduria (PA), cobalamin A (CblA), and cobalamin C (CblC) deficiencies. Neutrophil gelatinase-associated lipocalin (NGAL), calprotectin (CLP), kidney injury molecule-1 (KIM-1), dickkopf-3 (DKK-3), albumin and beta-2-microglobulin (B2MG) in urine, as well as cystatin C (CysC) in serum were quantified. In Mut0 patients, mean concentrations of B2MG, KIM-1, and DKK-3 were elevated compared with healthy controls, all markers indicative of proximal tubule damage. In PA patients, mean B2MG, albumin, and CLP were elevated, indicating signs of proximal tubule and glomerulus damage and inflammation. In CblC patients, mean B2MG, NGAL, and CLP were increased, and considered as markers for proximal and distal tubule damage and inflammation. B2MG, was elevated in all three diseases, and correlated with DKK-3 in Mut0 /CblA and with eGFR(CysC) and KIM-1 in PA patients, respectively. None of the markers were elevated in CblA patients. Significant deterioration of kidney function, as determined by steady increase in CysC concentrations was noted in seven patients within the observation period. None of the investigated biomarker profiles showed a clear increase or added value for early detection. In conclusion, we identified disease-specific biomarker profiles for inflammation, tubular, and proximal damage in the urine of Mut0 , PA, and CblC patients. Whether these biomarkers can be used for early detection of CKD requires further investigation, as significant kidney function deterioration was observed in only a few patients.

Carnosinase-1 Knock-Out Reduces Kidney Fibrosis in Type-1 Diabetic Mice on High Fat Diet.

Carnosine and anserine supplementation markedLy reduce diabetic nephropathy in rodents. The mode of nephroprotective action of both dipeptides in diabetes, via local protection or improved systemic glucose homeostasis, is uncertain. Global carnosinase-1 knockout mice (Cndp1-KO) and wild-type littermates (WT) on a normal diet (ND) and high fat diet (HFD) (n = 10/group), with streptozocin (STZ)-induced type-1 diabetes (n = 21-23/group), were studied for 32 weeks. Independent of diet, Cndp1-KO mice had 2- to 10-fold higher kidney anserine and carnosine concentrations than WT mice, but otherwise a similar kidney metabolome; heart, liver, muscle and serum anserine and carnosine concentrations were not different. Diabetic Cndp1-KO mice did not differ from diabetic WT mice in energy intake, body weight gain, blood glucose, HbA1c, insulin and glucose tolerance with both diets, whereas the diabetes-related increase in kidney advanced glycation end-product and 4-hydroxynonenal concentrations was prevented in the KO mice. Tubular protein accumulation was lower in diabetic ND and HFD Cndp1-KO mice, interstitial inflammation and fibrosis were lower in diabetic HFD Cndp1-KO mice compared to diabetic WT mice. Fatalities occurred later in diabetic ND Cndp1-KO mice versus WT littermates. Independent of systemic glucose homeostasis, increased kidney anserine and carnosine concentrations reduce local glycation and oxidative stress in type-1 diabetic mice, and mitigate interstitial nephropathy in type-1 diabetic mice on HFD.

High-Throughput Flavor Analysis and Mapping of Flavor Alterations Induced by Different Genotypes of Mentha by Means of UHPLC-MS/MS.

The demand for mint is increasing from year to year, and it is more important than ever to secure a sustainable and robust supply of such an important plant. The USDA mint core collection provides the basis for many researches worldwide regarding, e.g., sequencing, cytology, and disease resistances. A recently developed toolbox enables here for the first time the analysis of such a complex collection in terms of the aroma compound composition and the mapping of flavor alterations depending on taxonomy, environmental conditions, and growing stages by means of comprehensive liquid chromatography tandem mass spectrometry. Therefore, in this study, not only the aroma compound composition of 153 genotypes was characterized but it was also demonstrated that the composition varies depending on taxonomy and changes during the growth of the plant. Furthermore, it could be shown that greenhouse conditions have an enormous influence on the concentrations of aroma compounds.

Anserine and Carnosine Induce HSP70-Dependent H2S Formation in Endothelial Cells and Murine Kidney.

Anserine and carnosine have nephroprotective actions; hydrogen sulfide (H2S) protects from ischemic tissue damage, and the underlying mechanisms are debated. In view of their common interaction with HSP70, we studied possible interactions of both dipeptides with H2S. H2S formation was measured in human proximal tubular epithelial cells (HK-2); three endothelial cell lines (HUVEC, HUAEC, MCEC); and in renal murine tissue of wild-type (WT), carnosinase-1 knockout (Cndp1-KO) and Hsp70-KO mice. Diabetes was induced by streptozocin. Incubation with carnosine increased H2S synthesis capacity in tubular cells, as well as with anserine in all three endothelial cell lines. H2S dose-dependently reduced anserine/carnosine degradation rate by serum and recombinant carnosinase-1 (CN1). Endothelial Hsp70-KO reduced H2S formation and abolished the stimulation by anserine and could be restored by Hsp70 transfection. In female Hsp70-KO mice, kidney H2S formation was halved. In Cndp1-KO mice, kidney anserine concentrations were several-fold and sex-specifically increased. Kidney H2S formation capacity was increased 2-3-fold in female mice and correlated with anserine and carnosine concentrations. In diabetic Cndp1-KO mice, renal anserine and carnosine concentrations as well as H2S formation capacity were markedly reduced compared to non-diabetic Cndp1-KO littermates. Anserine and carnosine induce H2S formation in a cell-type and Hsp70-specific manner within a positive feedback loop with CN1.

Quantitative Mapping of Flavor and Pharmacologically Active Compounds in European Licorice Roots (Glycyrrhiza glabra L.) in Response to Growth Conditions and Arbuscular Mycorrhiza Symbiosis.

Application of a sensitive UHPLC-MS/MSMRM method enabled the simultaneous quantitation of 23 sweet-, licorice-, and bitter-tasting saponins in Glycyrrhiza glabra L., Glycyrrhiza uralensis Fisch., different licorice plants and root compartments, processed licorice, as well as different Glycyrrhiza spp. The combination of quantitative data with sweet, licorice, and bitter taste thresholds led to the determination of dose-over-threshold factors to elucidate the sweet, licorice, and bitter impact of the individual saponins with and without mycorrhiza symbiosis to evaluate the licorice root quality. Aside from glycyrrhizin (1), which is the predominant sweet- and licorice-tasting saponin in all licorice samples, 20 out of 22 quantitated saponins contributed to the taste profile of licorice roots. Next to sweet-/licorice-tasting glycyrrhizin (1), 24-hydroxy-glycyrrhizin (9), 30-hydroxy-glycyrrhizin (11), and 11-deoxo-24-hydroxy-glycyrrhizin (14) as well as licorice tasting saponins 20α-galacturonic acid glycyrrhizin (17), 24-hydroxy-20α-glycyrrhizin (21), and 11-deoxo-glycyrrhizin (12) were determined as key contributors to licorice root's unique taste profile. A quantitative comparison of 23 saponins as well as 28 polyphenols between licorice roots inoculated with arbuscular mycorrhiza fungi and controls showed that important taste-mediating saponins were increased in mycorrhizal roots, and these alterations depended on the growth substrate and the level of phosphate fertilization.

A Novel UPLC-MS/MS Method Identifies Organ-Specific Dipeptide Profiles.

BACKGROUND: Amino acids have a central role in cell metabolism, and intracellular changes contribute to the pathogenesis of various diseases, while the role and specific organ distribution of dipeptides is largely unknown. METHOD: We established a sensitive, rapid and reliable UPLC-MS/MS method for quantification of 36 dipeptides. Dipeptide patterns were analyzed in brown and white adipose tissues, brain, eye, heart, kidney, liver, lung, muscle, sciatic nerve, pancreas, spleen and thymus, serum and urine of C57BL/6N wildtype mice and related to the corresponding amino acid profiles. RESULTS: A total of 30 out of the 36 investigated dipeptides were detected with organ-specific distribution patterns. Carnosine and anserine were most abundant in all organs, with the highest concentrations in muscles. In liver, Asp-Gln and Ala-Gln concentrations were high, in the spleen and thymus, Glu-Ser and Gly-Asp. In serum, dipeptide concentrations were several magnitudes lower than in organ tissues. In all organs, dipeptides with C-terminal proline (Gly-Pro and Leu-Pro) were present at higher concentrations than dipeptides with N-terminal proline (Pro-Gly and Pro-Leu). Organ-specific amino acid profiles were related to the dipeptide profile with several amino acid concentrations being related to the isomeric form of the dipeptides. Aspartate, histidine, proline and serine tissue concentrations correlated with dipeptide concentrations, when the amino acids were present at the C- but not at the N-terminus. CONCLUSION: Our multi-dipeptide quantification approach demonstrates organ-specific dipeptide distribution. This method allows us to understand more about the dipeptide metabolism in disease or in healthy state.

A high throughput toolbox for comprehensive flavor compound mapping in mint.

Essential oils of the genus Mentha are extensively used as flavor ingredients in the industry. To overcome the time consuming and laborious traditional flavor analysis, a new quick, high-throughput toolbox based on a bead-beater homogenization followed by a UHPLC-MS/MS analysis has been developed and validated. While terpenes could be directly detected using atmospheric pressure chemical ionization (APCI), carbonyl compounds and alcohols required derivatization by 3-nitrophenylhydrazine (3-NPH) and glycidyltrimethylammonium chloride (GTMA) to ensure sufficient sensitivity for analysis of a single leaf. Using this approach, in total, 59 flavor-active metabolites representing the characteristic flavor of mint were quantified in leaves as well as in distilled oils using fast and robust UHPLC-MS/MS methods. The application of this toolbox enables a mapping of key pathways of mint flavor biosynthesis and can therefore support extensive breeding studies and the monitoring of chemosensate changes, depending on factors such as growth stages and environmental conditions.

Hydrogen Sulfide and Carnosine: Modulation of Oxidative Stress and Inflammation in Kidney and Brain Axis.

Emerging evidence indicates that the dysregulation of cellular redox homeostasis and chronic inflammatory processes are implicated in the pathogenesis of kidney and brain disorders. In this light, endogenous dipeptide carnosine (ß-alanyl-L-histidine) and hydrogen sulfide (H2S) exert cytoprotective actions through the modulation of redox-dependent resilience pathways during oxidative stress and inflammation. Several recent studies have elucidated a functional crosstalk occurring between kidney and the brain. The pathophysiological link of this crosstalk is represented by oxidative stress and inflammatory processes which contribute to the high prevalence of neuropsychiatric disorders, cognitive impairment, and dementia during the natural history of chronic kidney disease. Herein, we provide an overview of the main pathophysiological mechanisms related to high levels of pro-inflammatory cytokines, including interleukin-1ß (IL-1ß), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and neurotoxins, which play a critical role in the kidney-brain crosstalk. The present paper also explores the respective role of H2S and carnosine in the modulation of oxidative stress and inflammation in the kidney-brain axis. It suggests that these activities are likely mediated, at least in part, via hormetic processes, involving Nrf2 (Nuclear factor-like 2), Hsp 70 (heat shock protein 70), SIRT-1 (Sirtuin-1), Trx (Thioredoxin), and the glutathione system. Metabolic interactions at the kidney and brain axis level operate in controlling and reducing oxidant-induced inflammatory damage and therefore, can be a promising potential therapeutic target to reduce the severity of renal and brain injuries in humans.

Fatal outcome after heart surgery in PMM2-CDG due to a rare homozygous gene variant with double effects.

Variants in Phosphomannomutase 2 (PMM2) lead to PMM2-CDG, the most frequent congenital disorder of glycosylation (CDG). We here describe the disease course of a ten-month old patient who presented with the classical PMM2-CDG symptoms as cerebellar hypoplasia, retinitis pigmentosa, seizures, short stature, hepato- and splenomegaly, anaemia, recurrent vomiting and inverted mamillae. A severe form of tetralogy of Fallot was diagnosed and corrective surgery was performed at the age of 10 months. At the end of the cardiopulmonary bypass, a sudden oedematous reaction of the myocardium accompanied by biventricular pump failure was observed immediately after heparin antagonization with protamine sulfate. The patient died seven days after surgery, since myocardial function did not recover on ECMO support. We here describe the first patient carrying the homozygous variant g.18313A > T in the PMM2 gene (NG_009209.1) that either can lead to c.394A > T (p.I132F) or even loss of 100 bp due to exon 5 skipping (c.348_447del; p.G117Rfs*4) which is comparable to a null allele. Proliferation and doubling time of the patient's fibroblasts were affected. In addition, we show that the induction of cellular stress by elevating the cell culture temperature to 40 °C led to a decrease of the patients' PMM2 transcript as well as PMM2 protein levels and subsequently to a significant loss of residual activity. We assume that metabolic stressful processes occurring after cardiac surgery led to the drop of the patient's PMM activity below a life-sustaining niveau which paved the way for the fatal outcome.

A Global Cndp1-Knock-Out Selectively Increases Renal Carnosine and Anserine Concentrations in an Age- and Gender-Specific Manner in Mice.

Carnosinase 1 (CN1) is encoded by the Cndp1 gene and degrades carnosine and anserine, two natural histidine-containing dipeptides. In vitro and in vivo studies suggest carnosine- and anserine-mediated protection against long-term sequelae of reactive metabolites accumulating, e.g., in diabetes mellitus. We have characterized the metabolic impact of CN1 in 11- and 55-week-old Cndp1-knockout (Cndp1-KO) mice and litter-matched wildtypes (WT). In Cndp1-KO mice, renal carnosine and anserine concentrations were gender-specifically increased 2- to 9-fold, respectively in the kidney and both most abundant in the renal cortex, but remained unchanged in all other organs and in serum. Renal oxidized/reduced glutathione concentrations, renal morphology and function were unaltered. In Cndp1-KO mice at week 11, renal asparagine, serine and glutamine levels and at week 55, renal arginine concentration were reduced. Renal heat-shock-protein 70 (Hspa1a/b) mRNA declined with age in WT but not in Cndp1-KO mice, transcription factor heat-shock-factor 1 was higher in 55-week-old KO mice. Fasting blood glucose concentrations decreased with age in WT mice, but were unchanged in Cndp1-KO mice. Blood glucose response to intraperitoneal insulin was gender- but not genotype-dependent, the response to intraperitoneal glucose injection was similar in all groups. A global Cndp1-KO selectively, age- and gender-specifically, increases renal carnosine and anserine concentrations, alters renal amino acid- and HSP70 profile and modifies systemic glucose homeostasis. Increase of the natural occurring carnosine and anserine levels in the kidney by modulation of CN1 represents a promising therapeutic approach to mitigate or prevent chronic kidney diseases such as diabetic nephropathy.

Carnosine Activates Cellular Stress Response in Podocytes and Reduces Glycative and Lipoperoxidative Stress.

Carnosine improves diabetic complications, including diabetic nephropathy, in in vivo models. To further understand the underlying mechanism of nephroprotection, we studied the effect of carnosine under glucose-induced stress on cellular stress response proteins in murine immortalized podocytes, essential for glomerular function. High-glucose stress initiated stress response by increasing intracellular heat shock protein 70 (Hsp70), sirtuin-1 (Sirt-1), thioredoxin (Trx), glutamate-cysteine ligase (gamma-glutamyl cysteine synthetase; γ-GCS) and heme oxygenase-1 (HO-1) in podocytes by 30-50% compared to untreated cells. Carnosine (1 mM) also induced a corresponding upregulation of these intracellular stress markers, which was even more prominent compared to glucose for Hsp70 (21%), γ-GCS and HO-1 (13% and 20%, respectively; all p < 0.001). Co-incubation of carnosine (1 mM) and glucose (25 mM) induced further upregulation of Hsp70 (84%), Sirt-1 (52%), Trx (35%), γ-GCS (90%) and HO-1 (73%) concentrations compared to untreated cells (all p < 0.001). The glucose-induced increase in 4-hydroxy-trans-2-nonenal (HNE) and protein carbonylation was reduced dose-dependently by carnosine by more than 50% (p < 0.001). Although podocytes tolerated high carnosine concentrations (10 mM), high carnosine levels only slightly increased Trx and γ-GCS (10% and 19%, respectively, compared to controls; p < 0.001), but not Hsp70, Sirt-1 and HO-1 proteins (p not significant), and did not modify the glucose-induced oxidative stress response. In podocytes, carnosine induced cellular stress tolerance and resilience pathways and was highly effective in reducing high-glucose-induced glycative and lipoperoxidative stress. Carnosine in moderate concentrations exerted a direct podocyte molecular protective action.

Carnosine and Diabetic Nephropathy.

Diabetic Nephropathy (DN) is a major complication in patients with type 1 or type 2 diabetes and represents the leading cause of end-stage renal disease. Novel therapeutic approaches are warranted. In view of a polymorphism in the carnosinase 1 gene CNDP1, resulting in reduced carnosine degradation activity and a significant DN risk reduction, carnosine (ß-alanyl-L-histidine) has gained attention as a potential therapeutic target. Carnosine has anti-inflammatory, antioxidant, anti-glycation and reactive carbonyl quenching properties. In diabetic rodents, carnosine supplementation consistently improved renal histology and function and in most studies, also glucose metabolism. Even though plasma half-life of carnosine in humans is short, first intervention studies in (pre-) diabetic patients yielded promising results. The precise molecular mechanisms of carnosine mediated protective action, however, are still incompletely understood. This review highlights the recent knowledge on the role of the carnosine metabolism in DN.

Formation of 3-hydroxyglutaric acid in glutaric aciduria type I: in vitro participation of medium chain acyl-CoA dehydrogenase.

3-Hydroxyglutaric acid (3-OH-GA) in urine has been identified as the most reliable diagnostic marker for glutaric aciduria type I (GA I). We showed that hydratation of glutaconyl-CoA to 3-hydroxyglutaryl-CoA, which is subsequently hydrolyzed to 3-OH-GA, is efficiently catalyzed by 3-methylglutaconyl-CoA hydratase (3-MGH). We have now investigated whether mitochondrial acyl-CoA-dehydrogenases can convert glutaryl-CoA to glutaconyl-CoA. Short-chain acyl-CoA dehydrogenase (SCAD), medium-chain acyl-CoA dehydrogenase (MCAD), and long-chain acyl-CoA dehydrogenase (LCAD) accepted glutaryl-CoA as a substrate. The highest k cat of glutaryl-CoA was found for MCAD (0.12 ± 0.01 second-1) and was about 26-fold and 52-fold higher than those of LCAD and SCAD, respectively. The turnover of MCAD for glutaryl-CoA was about 1.5% of that of its natural substrate octanoyl-CoA. Despite high K m (above 600 µM) and low turnover rate, the oxidation of glutaryl-CoA by MCAD in combination with 3-MGH could explain the urinary concentration of 3-OH-GA in GA I patients.

Novel variants and clinical symptoms in four new ALG3-CDG patients, review of the literature, and identification of AAGRP-ALG3 as a novel ALG3 variant with alanine and glycine-rich N-terminus.

ALG3-CDG is one of the very rare types of congenital disorder of glycosylation (CDG) caused by variants in the ER-mannosyltransferase ALG3. Here, we summarize the clinical, biochemical, and genetic data of four new ALG3-CDG patients, who were identified by a type I pattern of serum transferrin and the accumulation of Man5 GlcNAc2 -PP-dolichol in LLO analysis. Additional clinical symptoms observed in our patients comprise sensorineural hearing loss, right-descending aorta, obstructive cardiomyopathy, macroglossia, and muscular hypertonia. We add four new biochemically confirmed variants to the list of ALG3-CDG inducing variants: c.350G>C (p.R117P), c.1263G>A (p.W421*), c.1037A>G (p.N346S), and the intron variant c.296+4A>G. Furthermore, in Patient 1 an additional open-reading frame of 141 bp (AAGRP) in the coding region of ALG3 was identified. Additionally, we show that control cells synthesize, to a minor degree, a hybrid protein composed of the polypeptide AAGRP and ALG3 (AAGRP-ALG3), while in Patient 1 expression of this hybrid protein is significantly increased due to the homozygous variant c.160_196del (g.165C>T). By reviewing the literature and combining our findings with previously published data, we further expand the knowledge of this rare glycosylation defect.