Kidney amino acid transport

Near complete reabsorption of filtered amino acids is a main specialized transport function of the kidney proximal tubule. This evolutionary conserved task is carried out by a subset of luminal and basolateral transporters that together form the transcellular amino acid transport machinery similar to that of small intestine. A number of other amino acid transporters expressed in the basolateral membrane of proximal kidney tubule cells subserve either specialized metabolic functions, such as the production of ammonium, or are part of the cellular housekeeping equipment. A new finding is that the luminal Na+-dependent neutral amino acid transporters of the SLC6 family require an associated protein for their surface expression as shown for the Hartnup transporter B0AT1 (SLC6A19) and suggested for the l-proline transporter SIT1 (IMINOB, SLC6A20) and for B0AT3 (XT2, SLC6A18). This accessory subunit called collectrin (TMEM27) is homologous to the transmembrane anchor region of the renin-angiotensin system enzyme ACE2 that we have shown to function in small intestine as associated subunit of the luminal SLC6 transporters B0AT1 and SIT1. Some mutations of B0AT1 differentially interact with these accessory subunits, providing an explanation for differential intestinal phenotypes among Hartnup patients. The basolateral efflux of numerous amino acids from kidney tubular cells is mediated by heteromeric amino acid transporters that function as obligatory exchangers. Thus, other transporters within the same membrane need to mediate the net efflux of exchange substrates, controlling thereby the net basolateral amino transport and thus the intracellular amino acid concentratio

Tenogenic differentiation protocol in xenogenic-free media enhances tendon-related marker expression in ASCs

Adipose-derived stem cells (ASCs) are multipotent and immune-privileged mesenchymal cells, making them ideal candidates for therapeutic purposes to manage tendon disorders. Providing safe and regulated cell therapy products to patients requires adherence to good manufacturing practices. To this aim we investigated the in vitro tenogenic differentiation potential of ASCs using a chemically defined serum-free medium (SF) or a xenogenic-free human pooled platelet lysate medium (hPL) suitable for cell therapy and both supplemented with CTGF, TGFβ-3, BMP-12 and ascorbic acid (AA) soluble factors. Human ASCs were isolated from 4 healthy donors and they were inducted to differentiate until 14 days in both hPL and SF tenogenic media (hPL-TENO and SF-TENO). Cell viability and immunophenotype profile were analysed to evaluate mesenchymal stem cell (MSC) characteristics in both xenogenic-free media. Moreover, the expression of stemness and tendon-related markers upon cell differentiation by RT-PCR, protein staining and cytofluorimetric analysis were also performed. Our results showed the two xenogenic-free media well support cell viability of ASCs and maintain their MSC nature as demonstrated by their typical immunophenototype profile and by the expression of NANOG, OCT4 and Ki67 genes. Moreover, both hPL-TENO and SF-TENO expressed significant high levels of the tendon-related genes SCX, COL1A1, COL3A1, COMP, MMP3 and MMP13 already at early time points in comparison to the respective controls. Significant up-regulations in scleraxis, collagen and tenomodulin proteins were also demonstrated at in both differentiated SF and hPL ASCs. In conclusion, we demonstrated firstly the feasibility of both serum and xenogenic-free media tested to culture ASCs moving forward the GMP-compliant approaches for clinical scale expansion of human MSCs needed for therapeutical application of stem cells. Moreover, a combination of CTGF, BMP-12, TGFβ3 and AA factors strongly and rapidly induce human ASCs to differentiate into tenocyte-like cells

Spheres Derived from Lung Adenocarcinoma Pleural Effusions: Molecular Characterization and Tumor Engraftment

Malignant pleural effusions (MPEs) could represent an excellent source to culture a wide variety of cancer cells from different donors. In this study, we set up culture conditions for cancer cells deriving from MPEs of several patients affected by the most frequent form of lung cancer, namely the subset of non small cell lung cancers (NSCLC) classified as Lung Adenocarcinomas (AdenoCa) which account for approximately 40% of lung cancer cases. AdenoCa malignant pleural effusions gave rise to in vitro cultures both in adherent and/or in spheroid conditions in almost all cases analyzed. We characterized in greater detail two samples which showed the most efficient propagation in vitro. In these samples we also compared gene profiles of spheroid vs adherent cultures and identified a set of differentially expressed genes. Finally we achieved efficient tumor engraftment in recipient NOD/SCID mice, also upon inoculation of small number of cells, thus suggesting indirectly the presence of tumor initiating cells

Amino Acids: Basolateral efflux and extracellular homeostasis control in vivo

In all living organisms, amino acids (AA) are essential building blocks, metabolites and signaling molecules. To reach their site of action AA need to pass through polar epithelial cells. Because AA cannot freely diffuse through the cell membrane, specific AA carriers ensure their transport across the apical and the basolateral membrane. Despite numerous flux studies and huge progress in the identification and characterization of those carrier proteins, AA transport across the basolateral membrane remains not fully understood. The best characterized basolateral transporters in the small intestine and kidney proximal tubule are the obligatory exchanger (antiporter) Lat2-4F2hc (Slc7a8) and y+Lat1-4F2hc (Slc7a7). Both require the presence of a "one-way transport" (uniporter) to achieve a net AA efflux. TAT1 (Slc16a10) and LAT4 (Slc43a2) fulfill this requirement with different substrate selectivities: TAT1 facilitates the diffusion of aromatic AA, whereas Lat4 transports branched chain AA, aromatic AA, Met and Pro. The functional cooperation between TAT1 and Lat2-4F2hc has previously been shown using the X. laevis oocyte expression system (Ramadan, Camargo et al. 2007). Furthermore, Lat4 could substitute for TAT1 function. Indeed, TAT1 shows the same localization as LAT2-4F2hc in epithelial cells and is further present in muscle sarcolemma and perivenous hepatocytes. The localization of Lat4 is still unknown, whereas the expression of the two uniporters overlaps only partially. The question addressed in this dissertation is: what is the function of TAT1 and Lat4 in vivo? Using global knock out mouse models we have examined the effect of the absence of the two uniporters on the AA homeostasis and epithelial transport. By inducing specific aminoaciduria in TAT1 defective mice (tat1-/-) under high protein diet, we could confirm the functional collaboration between TAT1 and LAT2-4F2hc in vivo. Furthermore, tat1-/- showed elevated aromatic AA in plasma, skeletal muscles and kidney, but not in liver. Thus, the absence of TAT1 prevents the AA balancing between plasma and liver and the consequent regulation of AA homeostasis. By means of everted gut sacs and microSPECT/CT imaging, we could show that the impairement of AA epithelial transport was caused by an intracellular accumulation of the AA substrates. The mild phenotype of tat1-/- strongly suggests a compensatory role of Lat4, which is also functionally redundant in the oocyte. In fact, Lat4 defective mice (lat4-/-) prematurely died within the first five days of life. After birth, the normal size, skin color and behavior of lat4-/- pups excluded possible prenatal impairments. Normal suckling behavior further excludes possible severe neurological disability. The reduced growth recorded between 24 and 48 hours indicated a disorder in food intake or in the reabsorption. Further investigations should aim to decipher the cause of death. In conclusion, this study analyzes the function of two basolateral uniporters for the first time in vivo. We show that the basolateral transport machinery is still functional in the absence of TAT1, whereas it is impaired in the absence of Lat4. Furthermore, we have shown a new important role of TAT1 in the regulation of the aromatic AA concentrations by the coupling of liver cells and blood plasma. Further research should aim to better characterize the in vivo interplay between AA transporters by the study of combinatorial knock out mice. Finally, the results of the different protein rich diets and their influence on the urinary pattern open new questions about the regulation of AA transporters in vivo

Online Black-box Failure Prediction for Mission Critical Distributed Systems

Abstract—Mission critical distributed systems are typically constructed out of many different software components strictly interacting with each other. Due to the inherent complexity of those systems and to the asynchrony of the interactions, they can exhibit anomalies that can lead to failures and, in some cases, to an abrupt block of the system, even though the software has been extensively tested. To avoid these failures and blocks, it is necessary to monitor at run time the system in order to discover such anomalies, thus predicting the occurrence of failures in advance. At this time, in order to minimize system damages appropriate measures can be taken by a system manager before the actual failure occurs. This paper introduces a novel approach to failure prediction for mission critical distributed systems that has the distinctive features to be (i) black-box: no knowledge of applications ’ internal

Processing Adipose Tissue Samples in a GMP Environment Standardizes the Use of SVF in Cell Therapy Treatments: Data on 302 Patients

Stromal vascular fraction (SVF) cells, together with adipose-derived mesenchymal stem cells, are becoming the tool of choice for many clinical applications. Currently, nearly 200 clinical trials are running worldwide to prove the efficacy of this cell type in treating many diseases and pathological conditions. To reach the goals of cell therapies and produce ATMPs as drugs for regenerative medicine, it is necessary to properly standardize GMP processes and, thus, collection methods, transportation strategies, extraction protocols, and characterization procedures, without forgetting that all the tissues of the human body are characterized by a wide inter-individual variability which is genetically determined and acquired during life. Here, we compare 302 samples processed under GMP rules to exclude the influence of the operator and of the anatomical site of collection. The influence of variability in the ages and genders of patients, along with laboratory parameters such as total cell number, cell viability, stem cell number, and other stromal vascular fraction cell subpopulations, has been compared. The results show that when the laboratory protocol is standardized, the variability of quantifiable cell parameters is widely statistically non-significant, meaning that we can take a further step toward standardized advanced cell therapy products

Upgrading Monocytes Therapy for Critical Limb Ischemia Patient Treatment: Pre-Clinical and GMP-Validation Aspects

Advanced cell therapy medicinal products (ATMP) are at the forefront of a new range of biopharmaceuticals. The use of ATMP has evolved and increased in the last decades, representing a new approach to treating diseases that are not effectively managed with conventional treatments. The standard worldwide recognized for drug production is the Good Manufacturing Practices (GMP), widely used in the pharma production of synthesized drugs but applying also to ATMP. GMP guidelines are worldwide recognized standards to manufacture medicinal products to guarantee high quality, safety, and efficacy. In this report, we describe the pre-clinical and the GMP upgrade of peripheral blood mononuclear cell (PBMC) preparation, starting from peripheral blood and ending up with a GMP-grade clinical product ready to be used in patients with critical limb ischemia (CLI). We also evaluated production in hypoxic conditions to increase PBMC functional activity and angiogenic potential. Furthermore, we extensively analyzed the storage and transport conditions of the final product as required by the regulatory body for ATMPs. Altogether, results suggest that the whole manufacturing process can be performed for clinical application. Peripheral blood collected by a physician should be transported at room temperature, and PBMCs should be isolated in a clean room within 8 h of venipuncture. Frozen cells can be stored in nitrogen vapors and thawed for up to 12 months. PBMCs resuspended in 5% human albumin solution should be stored and transported at 4 °C before injection in patients within 24 h to thawing. Hypoxic conditioning of PBMCs should be implemented for clinical application, as it showed a significant enhancement of PBMC functional activity, in particular with increased adhesion, migration, and oxidative stress resistance. We demonstrated the feasibility and the quality of a GMP-enriched suspension of monocytes as an ATMP, tested in a clean room facility for all aspects related to production in respect of all the GMP criteria that allow its use as an ATMP. We think that these results could ease the way to the clinical application of ATMPs

Essential amino acid transporter Lat4 (Slc43a2) is required for mouse development

KEY POINTS: Lat4 (Slc43a2) transports branched-chain amino acids, phenylalanine and methionine, and is expressed in kidney tubule and small intestine epithelial cells. Using a new knockout model as a negative control, it is shown that Lat4 is expressed at the basolateral side of small intestine enterocytes and kidney epithelial cells of the proximal tubule, thick ascending limb and distal convoluted tubule. In the Xenopus oocyte expression system, Lat4 is shown to function as a uniporter with symmetric intracellular and extracellular apparent affinities for phenylalanine. Mice lacking Lat4 display a slight intrauterine growth restriction, postnatal malnutrition and early death, presumably as a result of defective amino acid (re)absorption. These results demonstrate the crucial role that the uniporter Lat4 plays for amino acid transport across cellular barriers and mouse development. ABSTRACT: Amino acid (AA) uniporter Lat4 (Slc43a2) mediates facilitated diffusion of branched-chain AAs, methionine and phenylalanine, although its physiological role and subcellular localization are not known. We report that Slc43a2 knockout mice were born at expected Mendelian frequency but displayed an ∼10% intrauterine growth retardation and low amniotic fluid AAs, suggesting defective transplacental transport. Postnatal growth was strongly reduced, with premature death occurring within 9 days such that further investigations were made within 3 days of birth. Lat4 immunofluorescence showed a strong basolateral signal in the small intestine, kidney proximal tubule and thick ascending limb epithelial cells of wild-type but not Slc43a2 null littermates and no signal in liver and skeletal muscle. Experiments using Xenopus laevis oocytes demonstrated that Lat4 functioned as a symmetrical low affinity uniporter with a K0.5 of ∼5 mm for both in- and efflux. Plasma AA concentration was decreased in Slc43a2 null pups, in particular that of non-essential AAs alanine, serine, histidine and proline. Together with an increased level of plasma long chain acylcarnitines and a strong alteration of liver gene expression, this indicates malnutrition. Attempts to rescue pups by decreasing the litter size or by nutrients injected i.p. did not succeed. Radioactively labelled leucine but not lysine given per os accumulated in the small intestine of Slc43a2null pups, suggesting the defective transcellular transport of Lat4 substrates. In summary, Lat4 is a symmetrical uniporter for neutral essential AAs localizing at the basolateral side of (re)absorbing epithelia and is necessary for early nutrition and development