Cell surface receptors forgibbon apeleuke- miavirus (Glvr-1) andmarine amphotropic retrovirus (Ram-i) aredistinct butrelated proteins having multiple membrane- nning regions. Distant homology with aputative phosphate permease ofNeurospora crussa suggested that these receptors might serve transport functions. Byexpression inXenopus laevis oocytes andinmam l cells, wehaveidentified Glvr-1 andRam-iassodium-dependent phosphate symport- ers. Two-electrode voltage-camp analysis indicates netcation influx, gestngthatphosphate istransported withexcess sodium ions. Phosphate uptake wasreduced by>50%in mousefibroblasts expressing amphotropic envelope glycopro- tein, which binds toRam-i, indicating that Ram-iIsamajor phosphate transporter inthese cells. RNAanalysis shows wide butdistinct tissue distributions, withGlvr-1 expression being highest inbonemarrowandRam-i inheart. Overexpression of Ram-i severely reprsdGlvr-i synthesis infibroblasts, sug- gesting that transporter expression maybecontrolled bynet phosphate accumulation. Accordingly, depletion ofextracellu- larphosphate increased Ram-iandGlvr-i expression 3-to 5-fold. Theseresults suggest simple methods tomodulate retroviral receptor expression, withpossible applications to humangenetherapy. Keyissues invirology havebeenidentification ofcell-surface virus receptors, determination ofreceptor expression pat- terns, andelucidation oftheeffects ofinfection onthenormal functions ofthese molecules. Theseissues arecritical inthe caseofretroviruses because cells canbecomechronically infected, leading toreceptor down-modulation andsubse- quentderangements incellular functions. Forexample, the CD4receptor forhumanimmunodeficiency virus (HIV) is important forhelper T-cell function, andinterference with this normal function isanimportant aspect ofHIV-induced disease (1). Indeed, three genes ofHIV(env, nef, andvpu) caneachmediate CD4down-modulation (1-4). Whileseveral retroviral receptor cDNAshavebeeniso- lated (5-11), theonly functionally characterized receptors are theabove-mentioned CD4forHIVandasodium-indepen- dentcationic aminoacidtransporter forecotropic murine leukemia viruses (MLVs)(12, 13). Amongthecloned recep- tors with unknown functions arethose that mediate infections ofgibbon apeleukemia virus (GALV)andofmouseampho- tropic retrovirus (these receptors aretermed Glvr-1 and Ram-1,respectively) (7,10,11). Glvr-1 andRam-1are distinct proteins withmultiple hydrophobic potential mem- brane-spanning sequences. Although theycontain large un- related central domains, theyshare about60%overall se- quence identity andhaveabout 25%identity withaputative phosphate permease ofNeurospora crassa (10, 11,14,15). These data suggested that Glvr-1 andRam-1might betrans- porters; however, their solute specificities couldnotbe inferred because evenclosely related transport proteins often carry unrelated solutes (12, 13,16,17). Weidentified thetransporter activities ofGlvr-i andRam-1 byasystematic approach that involved expression ofthese proteins inXenopus laevis oocytes andmeasurement of transmembrane currents during exposure oftheoocytes to different salts andnutrients. Ourresults indicate that Glvr-1 andRam-1aresodium/phosphate symporters andthat these proteins provide amajor pathway forphosphate uptake into manymammalian cells. Inaddition, wehaveshownthat phosphate transport rates areaffected bothbyretroviral infection andbychanges inextracellular phosphate levels. MATERIALSANDMETHODS Cell Lines andRetroviral Vectors. Cell lines usedincluded 208Fratembryo fibroblasts (18), PA317cells (19), andNIH 3T3thymidine kinase-minus mouseembryo fibroblasts (20) andderivatives. Cells weregrowninDulbecco's minimal essential mediumwith10%o (vol/vol) fetal bovine serum. Retroviral vectors encoding neomycin phosphotransferase andratRam-1, humanGlvr-1, themouseecotropic receptor Rec-i, orhumanclotting factor IXweremadebycloning the respective cDNAsinto theretroviral vector LXSN(21). Vectors weretransfected into PA317retrovirus packaging cells (19), andtransiently produced virus washarvested after 2daysandwasusedtotransduce 208Fratfibroblasts as described (21) except fortheclotting factor IXvirus, which washarvested fromapreviously described stable vector- producing cell line (22). Transduced cells weregrowninthe presence ofG418toselect forcells expressing thevectors. Ion-Transport Assays inXenopus Oocytes Injected with Retrovirus Receptor RNAs.Thecoding regions oftherat Ram-i (10) andhumanGlvr-1 (7)cDNAswerecloned into pGEM-7Z(Promega) withtheir 5'endsadjacent totheSP6 promoter. FormRNA synthesis, theplasmids werelinear- ized andtranscribed withSP6polymerase inthepresence of m7G(5')ppp(5')G capsaccording tothemanufacturer's direc- tions (Pharmacia). Xenopus laevis oocytes wereinjected with 50nlofmRNA (1ng/nl) orwith anequal volume ofH20and wereincubated for4-6daysat17'C; thentwo-microelec- trode voltage-clamp recordings orradiolabel uptake assays

Cell-surface receptors forgibbon apeleukemia virus and amphotropic murineretrovirus areinducible sodium-dependent phosphate symporters

Oxidative stress has a critical role in the pathophysiology of several kidney diseases, and many complications of these diseases are mediated by oxidative stress, oxidative stress-related mediators, and inflammation. Several systemic diseases such as hypertension, diabetes mellitus, and hypercholesterolemia; infection; antibiotics, chemotherapeutics, and radiocontrast agents; and environmental toxins, occupational chemicals, radiation, smoking, as well as alcohol consumption induce oxidative stress in kidney. We searched the literature using PubMed, MEDLINE, and Google scholar with “oxidative stress, reactive oxygen species, oxygen free radicals, kidney, renal injury, nephropathy, nephrotoxicity, and induction”. The literature search included only articles written in English language. Letters or case reports were excluded. Scientific relevance, for clinical studies target populations, and study design, for basic science studies full coverage of main topics, are eligibility criteria for articles used in this paper.

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Induction of oxidative stress in kidney.

Urine is a valuable diagnostic medium and, with the discovery of urinary extracellular vesicles, is viewed as a dynamic bioactive fluid. Extracellular vesicles are lipid-enclosed structures that can be classified into three categories: exosomes, microvesicles (or ectosomes) and apoptotic bodies. This classification is based on the mechanisms by which membrane vesicles are formed: fusion of multivesicular bodies with the plasma membranes (exosomes), budding of vesicles directly from the plasma membrane (microvesicles) or those shed from dying cells (apoptotic bodies). During their formation, urinary extracellular vesicles incorporate various cell-specific components (proteins, lipids and nucleic acids) that can be transferred to target cells. The rigour needed for comparative studies has fueled the search for optimal approaches for their isolation, purification, and characterization. RNA, the newest extracellular vesicle component to be discovered, has received substantial attention as an extracellular vesicle therapeutic, and compelling evidence suggests that ex vivo manipulation of microRNA composition may have uses in the treatment of kidney disorders. The results of these studies are building the case that urinary extracellular vesicles act as mediators of renal pathophysiology. As the field of extracellular vesicle studies is burgeoning, this Review focuses on primary data obtained from studies of human urine rather than on data from studies of laboratory animals or cultured immortalized cells.

Isolation and characterization of urinary extracellular vesicles: implications for biomarker discovery

Nanomaterials application in electrochemical detection of heavy metals

Growing evidences suggest that the fibroblast growth factor/FGF receptor (FGF/FGFR) signaling has crucial roles in a multitude of processes during embryonic development and adult homeostasis by regulating cellular lineage commitment, differentiation, proliferation, and apoptosis of various types of cells. In this review, we provide a comprehensive overview of the current understanding of FGF signaling and its roles in organ development, injury repair, and the pathophysiology of spectrum of diseases, which is a consequence of FGF signaling dysregulation, including cancers and chronic kidney disease (CKD). In this context, the agonists and antagonists for FGF-FGFRs might have therapeutic benefits in multiple systems.

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FGF/FGFR signaling in health and disease

The heavy metal cadmium (Cd) is known to be a widespread environmental contaminant and a potential toxin that may adversely affect human health. Exposure is largely via the respiratory or gastrointestinal tracts; important non-industrial sources of exposure are cigarette smoke and food (from contaminated soil and water). The kidney is the main organ affected by chronic Cd exposure and toxicity. Cd accumulates in the kidney as a result of its preferential uptake by receptor-mediated endocytosis of freely filtered and metallothionein bound Cd (Cd-MT) in the renal proximal tubule. Internalised Cd-MT is degraded in endosomes and lysosomes, releasing free Cd(2+) into the cytosol, where it can generate reactive oxygen species (ROS) and activate cell death pathways. An early and sensitive manifestation of chronic Cd renal toxicity, which can be useful in individual and population screening, is impaired reabsorption of low molecular weight proteins (LMWP) (also a receptor-mediated process in the proximal tubule) such as retinol binding protein (RBP). This so-called 'tubular proteinuria' is a good index of proximal tubular damage, but it is not usually detected by routine clinical dipstick testing for proteinuria. Continued and heavy Cd exposure can progress to the clinical renal Fanconi syndrome, and ultimately to renal failure. Environmental Cd exposure may be a significant contributory factor to the development of chronic kidney disease, especially in the presence of other co-morbidities such as diabetes or hypertension; therefore, the sources and environmental impact of Cd, and efforts to limit Cd exposure, justify more attention.

Heavy metal poisoning: the effects of cadmium on the kidney

Inorganic phosphate (Pi) is an abundant element in the body and is essential for a wide variety of key biological processes. It plays an essential role in cellular energy metabolism and cell signalling, e.g. adenosine and guanosine triphosphates (ATP, GTP), and in the composition of phospholipid membranes and bone, and is an integral part of DNA and RNA. It is an important buffer in blood and urine and contributes to normal acid-base balance. Given its widespread role in almost every molecular and cellular function, changes in serum Pi levels and balance can have important and untoward effects. Pi homoeostasis is maintained by a counterbalance between dietary Pi absorption by the gut, mobilisation from bone and renal excretion. Approximately 85% of total body Pi is present in bone and only 1% is present as free Pi in extracellular fluids. In humans, extracellular concentrations of inorganic Pi vary between 0.8 and 1.2 mM, and in plasma or serum Pi exists in both its monovalent and divalent forms (H2PO4− and HPO42−). In the intestine, approximately 30% of Pi absorption is vitamin D regulated and dependent. To help maintain Pi balance, reabsorption of filtered Pi along the renal proximal tubule (PT) is via the NaPi-IIa and NaPi-IIc Na+-coupled Pi cotransporters, with a smaller contribution from the PiT-2 transporters. Endocrine factors, including, vitamin D and parathyroid hormone (PTH), as well as newer factors such as fibroblast growth factor (FGF)-23 and its coreceptor α-klotho, are intimately involved in the control of Pi homeostasis. A tight regulation of Pi is critical, since hyperphosphataemia is associated with increased cardiovascular morbidity in chronic kidney disease (CKD) and hypophosphataemia with rickets and growth retardation. This short review considers the control of Pi balance by vitamin D, PTH and Pi itself, with an emphasis on the insights gained from human genetic disorders and genetically modified mouse models.

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Physiological regulation of phosphate by vitamin D, parathyroid hormone (PTH) and phosphate (Pi)

Urine provides an attractive, non-invasive alternative to tissue, blood or other biofluid as a potential source of biomarkers of systemic and renal disease. The urinary protein content can be divided in two parts: soluble and solid-phase components; the solid-phase components can be subdivided into sediment precipitated with low-speed centrifugation and low-density nanometer-sized particles (vesicles <100 nm in diameter) precipitated by ultracentrifugation. In normal human adult urine, 48% of the total urinary protein excreted is contained in the sediment, 49% is soluble and the remaining 3% is in urinary vesicles [1]. Pisitkun et al. [2] were the first to describe these vesicles in human urine and called them ‘exosomes’; they went on to demonstrate the potential for exosomes as a starting material for biomarker discovery in urine. They chose the term exosomes because the vesicles originate from the membranes of internal multivesicular bodies that are released into the urine when the outer membrane of these bodies fuses with the apical plasma membrane of polarized epithelial cells [2]. Although the discovery of exosomes was intriguing, the question was why we should pay them any attention? Several recent studies have shown why exosomes are attractive for biomarker discovery. First, proteomics has shown that urinary exosomes are derived from all the epithelial cells lining the urinary tract, including glomerular podocytes and renal tubular cells from proximal and distal nephron segments [2]. The protein family commonly associated with exosomes is the tetraspanins, including CD9, CD63 and CD81. Second, other exosomal proteins directly represent the proteome of the source cells, for example, the presence of the sodium-potassium-chloride cotransporter type 2 (NKCC2), the sodium-chloride cotransporter (NCC) or the water channel aquaporin-2 (AQP2) reflects exosomes from the thick ascending limb of the loop of Henle, the distal convoluted and the collecting duct, respectively [2]. This property of exosomes is clinically useful as a biomarker not only of the nature of a renal disease, but also the site of renal injury [3]. For example, the abundance of the aldosterone-sensitive NCC was found to be higher in urinary exosomes of animal models of hyperaldosteronism and in patients with primary hyperaldosteronism [4]. Third, exosomes also contain transcription factors, messenger RNA (mRNA) and microRNA (miRNA), which have been shown to change in a disease [5–7]. Finally, the distal traffic of protein or RNA occurring after exosomes are released into the glomerular filtrate could potentially affect downstream cellular functions and be a novel mechanism of intra-renal signalling. Indeed, a recent study has demonstrated the ability of exosomes to mediate cell-to-cell communication along the nephron with functional AQP2 being transferred between cells [8]. The studies cited above provide a biological rationale for focussing more attention on urinary exosomes. However, as with many technical discoveries of this nature, there have been unresolved methodological problems. Since their initial discovery, the method of harvesting exosomes from various biological fluids, including urine, has relied on a two-step differential centrifugation process [2, 9, 10]. Although widely used, it is still uncertain whether differential centrifugation can isolate all urinary vesicles. This uncertainty was the basis of a recent and important study by Musante et al. [11] reported in the current issue of the journal. In their study, these investigators tried to optimize the process of vesicle isolation and yield, and, specifically, to define the properties of previously uncharacterized vesicles retained in the ultracentrifugation supernatant [11]. Using pooled second morning urines from six healthy volunteers, Musante et al. [11] set out to analyze whether urinary vesicles are still present in the original ultracentrifugation supernatant (‘the crude fraction’). To do so, they subjected the ultracentrifugation supernatant to overnight ammonium sulfate precipitation. A 10 000 g centrifugation step was then used to generate supernatant and pellet. This second supernatant was subjected to two further centrifugation steps and the resulting pellet was analyzed (‘the aqueous fraction’). The pellet obtained IN F O C U S

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Urinary vesicles: in splendid isolation

Proteases can increase the activity of the epithelial sodium channel (ENaC) by cleaving its α- or γ-subunit. However, evidence so far comes only from studies in vitro in either heterologous expression systems or isolated nephron segments. The present study has tested whether exposure to a luminal protease can alter sodium reabsorption along the rat collecting duct in vivo.

Protease stimulation of renal sodium reabsorption in vivo by activation of the collecting duct epithelial sodium channel (ENaC)

In the kidney, purinergic (P2) receptor-mediated ATP signaling has been shown to be an important local regulator of epithelial sodium transport. Appropriate sodium regulation is crucial for blood pressure (BP) control and disturbances in sodium balance can lead to hypo- or hypertension. Links have already been established between P2 receptor signaling and the development of hypertension, attributed mainly to vascular and/or inflammatory effects. A transgenic mouse model with deletion of the P2X4 receptor (P2X4-/- ) is known to have hypertension, which is thought to reflect endothelial dysfunction and impaired nitric oxide (NO) release. However, renal function in this model has not been characterized; moreover, studies in vitro have shown that the P2X4 receptor can regulate renal epithelial Na+ channel (ENaC) activity. Therefore, in the present study we investigated renal function and sodium handling in P2X4-/- mice, focusing on ENaC-mediated Na+ reabsorption. We confirmed an elevated BP in P2X4-/- mice compared with wild-type mice, but found that ENaC-mediated Na+ reabsorption is no different from wild-type and does not contribute to the raised BP observed in the knockout. However, when P2X4-/- mice were placed on a low sodium diet, BP normalized. Plasma aldosterone concentration tended to increase according to sodium restriction status in both genotypes; in contrast to wild-types, P2X4-/- mice did not show an increase in functional ENaC activity. Thus, although the increased BP in P2X4-/- mice has been attributed to endothelial dysfunction and impaired NO release, there is also a sodium-sensitive component.

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Grégory Jacquillet

Papers

Heavy metal poisoning: the effects of cadmium on the kidney

Physiological regulation of phosphate by vitamin D, parathyroid hormone (PTH) and phosphate (Pi)

Urinary vesicles: in splendid isolation

Protease stimulation of renal sodium reabsorption in vivo by activation of the collecting duct epithelial sodium channel (ENaC)

The renal and blood pressure response to low sodium diet in P2X4 receptor knockout mice