[Show abstract][Hide abstract] ABSTRACT: Intercalated cells (A-ICs) within the collecting duct of the kidney are critical for acid-base homeostasis. Here, we have shown that A-ICs also serve as both sentinels and effectors in the defense against urinary infec-tions. In a murine urinary tract infection model, A-ICs bound uropathogenic E. coli and responded by acidify-ing the urine and secreting the bacteriostatic protein lipocalin 2 (LCN2; also known as NGAL). A-IC–depen-dent LCN2 secretion required TLR4, as mice expressing an LPS-insensitive form of TLR4 expressed reduced levels of LCN2. The presence of LCN2 in urine was both necessary and sufficient to control the urinary tract infection through iron sequestration, even in the harsh condition of urine acidification. In mice lacking A-ICs, both urinary LCN2 and urinary acidification were reduced, and consequently bacterial clearance was limited. Together these results indicate that A-ICs, which are known to regulate acid-base metabolism, are also critical for urinary defense against pathogenic bacteria. They respond to both cystitis and pyelonephritis by delivering bacteriostatic chemical agents to the lower urinary system.
[Show abstract][Hide abstract] ABSTRACT: Kidney damage induces the expression of a myriad of proteins in the serum and in the urine. The function of these proteins in the sequence of damage and repair is now being studied in genetic models and by novel imaging techniques. One of the most intensely expressed proteins is lipocalin2, also called NGAL or Siderocalin. While this protein has been best studied by clinical scientists, only a few labs study its underlying metabolism and function in tissue damage. Structure-function studies, imaging studies and clinical studies have revealed that NGAL-Siderocalin is an endogenous antimicrobial with iron scavenging activity. This review discusses the "iron problem" of kidney damage, the tight linkage between kidney damage and NGAL-Siderocalin expression and the potential roles that NGAL-Siderocalin may serve in the defense of the urogenital system. This article is part of a Special Issue entitled: Cell Biology of Metals.
[Show abstract][Hide abstract] ABSTRACT: Many proteins have been proposed to act as surrogate markers of organ damage, yet for many candidates the essential biomarker characteristics that link the protein to the injured organ have not yet been described. We generated an Ngal reporter mouse by inserting a double-fusion reporter gene encoding luciferase-2 and mCherry (Luc2-mC) into the Ngal (Lcn2) locus. The Ngal-Luc2-mC reporter accurately recapitulated the endogenous message and illuminated injuries in vivo in real time. In the kidney, Ngal-Luc2-mC imaging showed a sensitive, rapid, dose-dependent, reversible, and organ- and cell-specific relationship with tubular stress, which correlated with the level of urinary Ngal (uNgal). Unexpectedly, specific cells of the distal nephron were the source of uNgal. Cells isolated from Ngal-Luc2-mC mice also revealed both the onset and the resolution of the injury, and the actions of NF-κB inhibitors and antibiotics during infection. Thus, imaging of Ngal-Luc2-mC mice and cells identified injurious and reparative agents that affect kidney damage.
Nature medicine 02/2011; 17(2):216-22. DOI:10.1038/nm.2290 · 27.36 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The lipocalins are secreted proteins that bind small organic molecules. Scn-Ngal (also known as neutrophil gelatinase associated lipocalin, siderocalin, lipocalin 2) sequesters bacterial iron chelators, called siderophores, and consequently blocks bacterial growth. However, Scn-Ngal is also prominently expressed in aseptic diseases, implying that it binds additional ligands and serves additional functions. Using chemical screens, crystallography and fluorescence methods, we report that Scn-Ngal binds iron together with a small metabolic product called catechol. The formation of the complex blocked the reactivity of iron and permitted its transport once introduced into circulation in vivo. Scn-Ngal then recycled its iron in endosomes by a pH-sensitive mechanism. As catechols derive from bacterial and mammalian metabolism of dietary compounds, the Scn-Ngal-catechol-Fe(III) complex represents an unforeseen microbial-host interaction, which mimics Scn-Ngal-siderophore interactions but instead traffics iron in aseptic tissues. These results identify an endogenous siderophore, which may link the disparate roles of Scn-Ngal in different diseases.
Nature Chemical Biology 08/2010; 6(8):602-9. DOI:10.1038/nchembio.402 · 13.00 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Developing organs require iron for a myriad of functions, but embryos deleted of the major adult transport proteins, transferrin or its receptor transferrin receptor1 (TfR1(-/-)), still initiate organogenesis, suggesting that non-transferrin pathways are important. To examine these pathways, we developed chimeras composed of fluorescence-tagged TfR1(-/-) cells and untagged wild-type cells. In the kidney, TfR1(-/-) cells populated capsule and stroma, mesenchyme and nephron, but were underrepresented in ureteric bud tips. Consistently, TfR1 provided transferrin to the ureteric bud, but not to the capsule or the stroma. Instead of transferrin, we found that the capsule internalized ferritin. Since the capsule expressed a novel receptor called Scara5, we tested its role in ferritin uptake and found that Scara5 bound serum ferritin and then stimulated its endocytosis from the cell surface with consequent iron delivery. These data implicate cell type-specific mechanisms of iron traffic in organogenesis, which alternatively utilize transferrin or non-transferrin iron delivery pathways.
[Show abstract][Hide abstract] ABSTRACT: The reduced folate carrier (RFC) and the proton-coupled folate transporter (PCFT) are ubiquitously expressed in normal and malignant mammalian tissues and in human solid tumor cell lines. This article addresses the extent to which PCFT contributes to transport of pemetrexed and to the activities of this and other antifolates relative to RFC at physiological pH. Either RFC or PCFT cDNA was stably transfected into a transporter-null HeLa cell variant to achieve activities similar to their endogenous function in wild-type HeLa cells. PCFT and RFC produced comparable increases in pemetrexed activity in growth medium with 5-formyltetrahydrofolate. However, PCFT had little or no effect on the activities of methotrexate, N-(5-[N-(3,4-dihydro-2-methyl-4-oxyquinazolin-6-ylmethyl)-N-methyl-amino]-2-thenoyl)-l-glutamic acid (raltitrexed, Tomudex; ZD1694), or N(alpha)-(4-amino-4-deoxypteroyl)-N(delta)-hemiphthaloyl-l-ornithine (PT523) in comparison with RFC irrespective of the folate growth source. PCFT, expressed at high levels in Xenopus laevis oocytes and in transporter-competent HepG2 cells, exhibited a high affinity for pemetrexed, with an influx K(m) value of 0.2 to 0.8 muM at pH 5.5. PCFT increased the growth inhibitory activity of pemetrexed, but not that of the other antifolates in HepG2 cells grown with 5-formyltetrahydrofolate at physiological pH. These findings illustrate the unique role that PCFT plays in the transport and pharmacological activity of pemetrexed. Because of the ubiquitous expression of PCFT in human tumors, and the ability of PCFT to sustain pemetrexed activity even in the absence of RFC, tumor cells are unlikely to become resistant to pemetrexed as a result of impaired transport because of the redundancy of these genetically distinct routes.
[Show abstract][Hide abstract] ABSTRACT: The human proton-coupled folate transporter (HsPCFT, SLC46A1) mediates intestinal absorption of folates and transport of folates into the liver, brain and other tissues. On Western blot, HsPCFT migrates as a broad band (~55 kDa), higher than predicted (~50 kDa) in cell lines. Western blot analysis required that membrane preparations not be incubated in the loading buffer above 50 degrees C to avoid aggregation of the protein. Treatment of membrane fractions from HsPCFT-transfected HeLa cells with peptidyl N-glycanase F, or cells with tunicamycin, resulted in conversion to a ~35 kDa species. Substitution of asparagine residues of two canonical glycosylation sites to glutamine, individually, yielded a ~47 kDa protein; substitution of both sites gave a smaller (~35 kDa) protein. Single mutants retained full transport activity; the double mutant retained a majority of activity. Transport function and molecular size were unchanged when the double mutant was hemagglutinin (HA) tagged at either the NH(2) or COOH terminus and probed with an anti-HA antibody excluding degradation of the deglycosylated protein. Wild-type or deglycosylated HsPCFT HA, tagged at amino or carboxyl termini, could only be visualized on the plasma membrane when HeLa cells were first permeabilized, consistent with the intracellular location of these domains.
[Show abstract][Hide abstract] ABSTRACT: Reduced derivatives of folic acid (folates) play a critical role in the development, function and repair of the CNS. However, the molecular systems regulating folate uptake and homeostasis in the central nervous system remain incompletely defined. Choroid plexus epithelial cells express high levels of folate receptor alpha (FRalpha) suggesting that the choroid plays an important role in CNS folate trafficking and maintenance of CSF folate levels. We have characterized 5-methyltetrahydrofolate (5-MTHF) uptake and metabolism by primary rat choroid plexus epithelial cells in vitro. Two distinct processes are apparent; one that is FRalpha dependent and one that is independent of the receptor. FRalpha binds 5-MTHF with high affinity and facilitates efficient uptake of 5-MTHF at low extracellular folate concentrations; a lower affinity FRalpha independent system accounts for increased folate uptake at higher concentrations. Cellular metabolism of 5-MTHF depends on the route of folate entry into the cell. 5-MTHF taken up via a non-FRalpha -mediated process is rapidly metabolized to folylpolyglutamates, whereas 5-MTHF that accumulates via FRalpha remains non-metabolized, supporting the hypothesis that FRalpha may be part of a pathway for transcellular movement of the vitamin. The proton-coupled folate transporter, proton-coupled folate transporter (PCFT), mRNA was also shown to be expressed in choroid plexus epithelial cells. This is consistent with the role we have proposed for proton-coupled folate transporter in FRalpha-mediated transport as the mechanism of export of folates from the endocytic compartment containing FRalpha.
Journal of Neurochemistry 04/2008; 104(6):1494-503. DOI:10.1111/j.1471-4159.2007.05095.x · 4.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: This laboratory recently identified a human gene that encodes a novel folate transporter [Homo sapiens proton-coupled folate transporter (HsPCFT); SLC46A1] required for intestinal folate absorption. This study focused on mouse (Mus musculus) PCFT (MmPCFT) and rat (Rattus norvegicus) PCFT (RnPCFT) and addresses their secondary structure, specificity, tissue expression, and regulation by dietary folates. Both rodent PCFT proteins traffic to the cell membrane with the NH(2)- and COOH-termini accessible to antibodies targeted to these domains only in permeabilized HeLa cells. This, together with computer-based topological analyses, is consistent with a model in which rodent PCFT proteins likely contain 12 transmembrane domains. Transport of [(3)H]folates was optimal at pH 5.5 and decreased with increasing pH due to an increase in K(m) and a decrease in V(max). At pH 7.0, folic acid and methotrexate influx was negligible, but there was residual (6S)5-methyltetrahydrofolate transport. Uptake of folates in PCFT-injected Xenopus oocytes was electrogenic and pH dependent. Folic acid influx K(m) values of MmPCFT and RnPCFT, assessed electrophysiologically, were 0.7 and 0.3 microM at pH 5.5 and 1.1 and 0.8 microM at pH 6.5, respectively. Rodent PCFTs were highly specific for monoglutamyl but not polyglutamyl methotrexate. MmPCFT mRNA was highly expressed in the duodenum, proximal jejunum, liver, and kidney with lesser expression in the brain and other tissues. MmPCFT protein was localized to the apical brush-border membrane of the duodenum and proximal jejunum. MmPCFT mRNA levels increased approximately 13-fold in the proximal small intestine in mice fed a folate-deficient vesus folate-replete diet, consistent with the critical role that PCFT plays in intestinal folate absorption.
[Show abstract][Hide abstract] ABSTRACT: Hereditary folate malabsorption (HFM) is a rare autosomal recessive disorder caused by impaired intestinal folate absorption and impaired folate transport into the central nervous system. Recent studies in 1 family revealed that the molecular basis for this disorder is a loss-of-function mutation in the PCFT gene encoding a proton-coupled folate transporter. The current study broadens the understanding of the spectrum of alterations in the PCFT gene associated with HFM in 5 additional patients. There was no racial, ethnic, or sex pattern. A total of 4 different homozygous mutations were detected in 4 patients; 2 heterozygous mutations were identified in the fifth patient. Mutations involved 4 of the 5 exons, all at highly conserved amino acid residues. A total of 4 of the mutated transporters resulted in a complete loss of transport function, primarily due to decreased protein stability and/or defects in membrane trafficking, while 2 of the mutated carriers manifested residual function. Folate transport at low pH was markedly impaired in transformed lymphocytes from 2 patients. These findings further substantiate the role that mutations in PCFT play in the pathogenesis of HFM and will make possible rapid diagnosis and treatment of this disorder in infants, and prenatal diagnosis in families that carry a mutated gene.
[Show abstract][Hide abstract] ABSTRACT: Folates are essential nutrients that are required for one-carbon biosynthetic and epigenetic processes. While folates are absorbed in the acidic milieu of the upper small intestine, the underlying absorption mechanism has not been defined. We now report the identification of a human proton-coupled, high-affinity folate transporter that recapitulates properties of folate transport and absorption in intestine and in various cell types at low pH. We demonstrate that a loss-of-function mutation in this gene is the molecular basis for hereditary folate malabsorption in a family with this disease. This transporter was previously reported to be a lower-affinity, pH-independent heme carrier protein, HCP1. However, the current study establishes that a major function of this gene product is proton-coupled folate transport required for folate homeostasis in man, and we have thus amended the name to PCFT/HCP1.