Tyrosine hydroxylase catalyses the hydroxylation of L-tyrosine to l-DOPA, the rate-limiting step in the synthesis of catecholamines. Mutations in the TH gene encoding tyrosine hydroxylase are associated with the autosomal recessive disorder tyrosine hydroxylase deficiency, which manifests phenotypes varying from infantile parkinsonism and DOPA-responsive dystonia, also termed type A, to complex encephalopathy with perinatal onset, termed type B. We generated homozygous Th knock-in mice with the mutation Th-p.R203H, equivalent to the most recurrent human mutation associated with type B tyrosine hydroxylase deficiency (TH-p.R233H), often unresponsive to l-DOPA treatment. The Th knock-in mice showed normal survival and food intake, but hypotension, hypokinesia, reduced motor coordination, wide-based gate and catalepsy. This phenotype was associated with a gradual loss of central catecholamines and the serious manifestations of motor impairment presented diurnal fluctuation but did not improve with standard l-DOPA treatment. The mutant tyrosine hydroxylase enzyme was unstable and exhibited deficient stabilization by catecholamines, leading to decline of brain tyrosine hydroxylase-immunoreactivity in the Th knock-in mice. In fact the substantia nigra presented an almost normal level of mutant tyrosine hydroxylase protein but distinct absence of the enzyme was observed in the striatum, indicating a mutation-associated mislocalization of tyrosine hydroxylase in the nigrostriatal pathway. This hypomorphic mouse model thus provides understanding on pathomechanisms in type B tyrosine hydroxylase deficiency and a platform for the evaluation of novel therapeutics for movement disorders with loss of dopaminergic input to the striatum.

Rapid and affordable detection of analytes is critical in diagnostic technologies, but current methods are typically expensive and unsuitable for field detection. Lipidic cubic phases are optically isotropic, transparent lyotropic liquid crystals (LC), containing highly confined water nanochannels in‐between percolating lipid bilayers following defined space groups. Due to this nanoconfinement, the water in these systems provides a unique environment for chemical and enzymatic reactions. Here, it is shown that during the in meso peroxidase enzymatic reaction, the converted product crystallizes within the mesophase domains, generating a detectable birefringence signal and a new general assay principle is presented for the detection of an unprecedented vast class of analytes using such birefringence as sole optical output signal. By exploiting bienzymatic cascade reactions or introducing an enzyme‐linked immunosorbent assay based on birefringence (Birefringent‐ELISA), this approach is used for real‐time detection of exemplary analytes, such as glucose and cholesterol, model pathogenic microorganisms, Escherichia coli, and viruses such as Ebola and HIV. It is also shown how the same technology enables the rapid, naked‐eye screening of malaria infection via in meso detection of hemozoin crystallites. This new technology is general and readily adaptable to the rapid detection of virtually any type of analyte, such as disease biomarkers, viruses, bacteria, and parasites.

The SLC36 family of transporters consists of four genes, two of which, SLC36A1 and SLC36A2, have been demonstrated to code for human proton-coupled amino acid transporters or hPATs. Here we report the characterization of the fourth member of the family, SLC36A4 or hPAT4, which when expressed in Xenopus laevis oocytes also encodes a plasma membrane amino acid transporter, but one that is not proton-coupled and has a very high substrate affinity for the amino acids proline and tryptophan. hPAT4 in Xenopus oocytes mediated sodium-independent, electroneutral uptake of [ 3 H]proline, with the highest rate of uptake when the uptake medium pH was 7.4 and an affinity of 3.13 M. Tryptophan was also an excellently transported substrate with a similarly high affinity (1.72 M). Other amino acids that inhibited [ 3 H]proline were isoleucine (K i 0.23 mM), glutamine (0.43 mM), methionine (0.44 mM), and alanine (1.48 mM), and with lower affinity, glycine, threonine, and cysteine (K i >5 mM for all). Of the amino acids directly tested for transport, only proline, tryptophan, and alanine showed significant uptake, whereas glycine and cysteine did not. Of the non-proteogenic amino acids and drugs tested, only sarcosine produced inhibition (K i 1.09 mM), whereas ␥-aminobutyric acid (GABA), ␤-alanine, L-Dopa, D-serine, and ␦-aminolevulinic acid were without effect on [ 3 H]proline uptake. This characterization of hPAT4 as a very high affinity/low capacity non-proton-coupled amino acid transporter raises questions about its physiological role, especially as the transport characteristics of hPAT4 are very similar to the Drosophila orthologue PATH, an amino acid "transceptor" that plays a role in nutrient sensing.

The metabolism of dietary proteins generates waste products that are excreted by the kidney, in particular nitrogen-containing urea, uric acid, ammonia, creatinine, and other metabolites such as phosphates, sulfates, and protons. Kidney adaptation includes an increase in renal plasma flow (RPF) and glomerular filtration rate (GFR) and represents a burden for diseased kidneys increasing the progression rate of CKD. The present study aimed at identifying potential differences between amino acid (AA) groups constituting dietary proteins regarding their impact on RPF, GFR, and CKD progression. We utilized the well-established 5/6 nephrectomy (5/6 Nx) CKD model in rats and submitted the animals for 5 weeks to either the control diet (18% casein protein) or to diets containing 8% casein supplemented with 10% of a mix of free amino acids, representing all or only a subset of the amino acids contained in casein. Whereas the RPF and GFR measured in free moving animals remained stable during the course of the diet in rats receiving the control mix, these parameters decreased in animals receiving the branched chain amino acid (BCAA) supplementation and increased in the ones receiving the aromatic amino acids (AAAs). In animals receiving essential amino acids (EAAs) containing both BCAAs and AAAs, there was only a small increase in RPF. The kidneys of the 5/6 Nx rats receiving the BCAA diet showed the strongest increase in smooth muscle actin and collagen mRNA expression as a result of higher level of inflammation and fibrosis. These animals receiving BCAAs also showed an increase in plasma free fatty acids pointing to a problem at the level of energy metabolism. In contrast, the animals under AAA diet showed an activation of AMPK and STAT3. Taken together, our results demonstrate that subsets of EAAs contained in dietary proteins, specifically BCAAs and AAAs, exert contrasting effects on kidney functional parameters and CKD progression.

BackgroundUninephrectomy (UNX) is performed for various reasons, including kidney cancer or donation. Kidneys being the main site of l‐arginine production in the body, we tested whether UNX mediated kidney mass reduction impacts l‐arginine metabolism and thereby nitric oxide production and blood pressure regulation in mice.Methods and ResultsIn a first series of experiments, we observed a significant increase in arterial blood pressure 8 days post‐UNX in female and not in male mice. Further experimental series were performed in female mice, and the blood pressure increase was confirmed by telemetry. l‐citrulline, that is used in the kidney to produce l‐arginine, was elevated post‐UNX as was also asymmetric dimethylarginine, an inhibitor of nitric oxide synthase that competes with l‐arginine and is a marker for renal failure. Interestingly, the UNX‐induced blood pressure increase was prevented by supplementation of the diet with 5% of the l‐arginine precursor, l‐citrulline. Because l‐arginine is metabolized in the kidney and other peripheral tissues by arginase‐2, we tested whether the lack of this metabolic pathway also compensates for decreased l‐arginine production in the kidney and/or for local nitric oxide synthase inhibition and consecutive blood pressure increase. Indeed, upon uninephrectomy, arginase‐2 knockout mice (Arg‐2−/−) neither displayed an increase in asymmetric dimethylarginine and l‐citrulline plasma levels nor a significant increase in blood pressure.ConclusionsUNX leads to a small increase in blood pressure that is prevented by l‐citrulline supplementation or arginase deficiency, 2 measures that appear to compensate for the impact of kidney mass reduction on l‐arginine metabolism.

With worldwide epidemic outbreaks occurring on a regular basis, there is urgent need to improve and establish new strategies toward rapid, simple, and affordable detection of target analytes. Birefringence generated from versatile lipid based liquid crystal cubic phase leads to a unique optical signal that can be used to detect analytes of medical interest, including bacteria, viruses, and parasites, as shown by R. Mezzenga and co‐workers on page 181.

Chronic kidney disease (CKD) affects over 10% of the population (Troidle, 2014) and is on the rise. An increase in the protein intake, one of the characteristics of a western‐type diet, is thought to have a detrimental effect by increasing the Renal Plasma Flow (RPF), inducing hyperfiltration and increasing acid load. In addition to these, the source of the protein i.e. animal or vegetal (Goraya and Wesson, 2016) and consequently its amino acid content, might also play a role in the progression of the disease. The actual mechanisms by which these proteins / amino acids lead to increased Glomerular Filtration Rate (GFR) and to kidney function deterioration is not well understood.In our experiments, we address the question of which amino acids, or groups of amino acids, might affect the progression of renal disease.To address this question, we utilized the well‐established 5/6th Nephrectomy (5/6th Nx) model. 5/6th Nx Wistar Han rats were randomly divided into groups, receiving either the control diet (18% protein), or one of different diets, in each case containing 8% protein supplemented with 10% amino acid (aa) mix (Essential aas, Non‐essential aas, Branched chain aas or all aas in the same proportion as in the protein mix). Both GFR and RPF were measured in free moving animals, GFR transcutaneously using FITC‐sinistrin, and RPF by using radiolabelled para‐aminohippurate (PAH). None of the modified diets caused any decrease in body weight, food or water consumption in the different groups. However, the pace of RPF and GFR alteration was diet‐dependent, with the slowest change in animals receiving the EAA diet, and the most dramatic one in the animals on BCAAs. The kidney of these BCAA receiving rats also showed the strongest increase in smooth muscle actin and collagen mRNA expression in their kidney, as expected for a higher level of inflammation and fibrosis. The SMA levels were increased 2‐fold and the collagen levels were increased more than 3‐fold compared to animals on the control diet. Taken together, these results suggest that high levels of BCAAs contained in the diet have a deleterious effect on the progression of CKD.Support or Funding InformationThis research/project was supported by the NCCR Kidney. CH funded by the Swiss National Science Foundation

L‐arginine plays an important role, acting as a precursor for a variety of physiologically important substances including citrulline, urea, ornithine, proline and nitric oxide and the kidney is a major site for its metabolism. Uninephrectomy (UNX) is observed to cause an increase in the size of the remnant kidney and compensation to some degree of the glomerular filtration rate. As very little is known about UNX‐induced effects on blood pressure control and expression levels of transporters and enzymes involved in arginine metabolism, we are testing in mice the hypothesis that renal mass reduction impacts on Arg metabolism and possibly thereby affects blood pressure control. Our results show that in contrast to sham operated controls, mice having undergone UNX display an increased systolic blood pressure (120 ± 2.14 vs. 112 ± 1.97 mmHg by tail cuff measurements, n = 18). This effect was more pronounced in females than males and observed also by telemetry. Plasma levels of asymmetric dimethyl arginine (ADMA), an inhibitor of NOS considered to be a good marker for renal disease, were increased in UNX animals, whereas the level of none of the proteinogenic amino acids was changed significantly. There were also no changes in the mRNA expression levels of Arg transporters and enzymes involved in arginine metabolism. Our observations suggest that UNX affects blood pressure and the effects are less pronounced in males, possibly due to a more important remnant kidney compensatory growth. Grant Funding Source: Supported by the Swiss NCCR Kidney.ch and the European Community's Seventh Framework Programme