Here we review the structural and functional properties of organic anion transporters (OAT1, OAT2, OAT3) and organic cation transporters (OCTN1, OCTN2, OCT1, OCT2, OCT3), some of which are involved in renal proximal tubular organic anion and cation secretion. These transporters share a predicted 12-transmembrane domain (TMD) structure with a large extracellular loop between TMD1 and TMD2, carrying potential N-glycosylation sites. Conserved amino acid motifs revealed a relationship to the sugar transporter family within the major facilitator superfamily. Following heterologous expression, most OATs transported the model anion p-aminohippurate (PAH). OAT1, but not OAT2, exhibited PAH-alpha-ketoglutarate exchange. OCT1-3 transported the model cations tetraethylammonium (TEA), N(1)-methylnicotinamide, and 1-methyl-4-phenylpyridinium. OCTNs exhibited transport of TEA and/or preferably the zwitterionic carnitine. Substrate substitution as well as cis-inhibition experiments demonstrated polyspecificity of the OATs, OCTs, and OCTN1. On the basis of comparison of the structurally closely related OATs and OCTs, it may be possible to delineate the binding sites for organic anions and cations in future experiments.

Background: Localization and function of the lipocalin-2/NGAL/24p3 receptor (24p3R) in the kidney are unknown. Results: 24p3R is expressed in apical plasma membranes of the distal nephron and mediates high-affinity protein endocytosis in renal cells. Conclusion: 24p3R contributes to protein endocytosis and nephrotoxicity in distal nephron segments. Significance: This is the first study to investigate localization and function of 24p3R in relevant epithelia.

Neural stem cells (NSCs) are potential sources for cell therapy of neurodegenerative diseases and for drug screening. Despite their potential benefits, ethical and practical considerations limit the application of NSCs derived from human embryonic stem cells (ES) or adult brain tissue. Thus, alternative sources are required to satisfy the criteria of ready accessibility, rapid expansion in chemically defined media and reliable induction to a neuronal fate. We isolated somatic stem cells from the human periodontium that were collected during minimally invasive periodontal access flap surgery as part of guided tissue regeneration therapy. These cells could be propagated as neurospheres in serum-free medium, which underscores their cranial neural crest cell origin. Culture in the presence of epidermal growth factor (EGF) and fibroblast growth factor-2 (FGF-2) under serum-free conditions resulted in large numbers of nestin-positive/Sox-2-positive NSCs. These periodontium-derived (pd) NSCs are highly proliferative and migrate in response to chemokines that have been described as inducing NSC migration. We used immunocytochemical techniques and RT-PCR analysis to assess neural differentiation after treatment of the expanded cells with a novel induction medium. Adherence to substrate, growth factor deprivation, and retinoic acid treatment led to the acquisition of neuronal morphology and stable expression of markers of neuronal differentiation by more than 90% of the cells. Thus, our novel method might provide nearly limitless numbers of neuronal precursors from a readily accessible autologous adult human source, which could be used as a platform for further experimental studies and has potential therapeutic implications.

Background/Aims: Renal secretion of organic anions is critically dependent on their basolateral uptake against the electrochemical gradient. Due to their localization, two transporters are likely involved, namely OAT1 and OAT3. While OAT1 as an exchanger clearly operates in the secretory direction, OAT3 in its previously supposed mode as a uniporter should move anionic substrates from cell to blood. It would thus dissipate gradients established by OAT1 of common OAT1/OAT3 substrates. In the present study we therefore reinvestigated the driving forces of human OAT3. Methods: The human OAT3 obtained from the Resource Center/Primary Database was made functional by site-directed mutagenesis. Using the Xenopus laevis oocyte expression system, hOAT3-mediated transport of estrone sulfate (ES) and dicarboxylates was assayed for cis-inhibition and/or trans-stimulation in both the uptake and efflux direction. Results: hOAT3-mediated efflux of glutarate (GA), can be significantly trans-stimulated by a variety of ions with high cis-inhibitory potency, including GA (282%), α-ketoglutarate (476%), p-aminohippurate (179%), and, most notably, urate (167%). Urate cis-inhibited ES uptake with an IC₅₀ close to normal serum urate concentrations. Conclusion: These data indicate that OAT3 does not represent a uniporter but operates as an organic ion%dicarboxylate exchanger similar to OAT1, and may mediate renal urate secretion.

The cDNA coding for a renal />-aminohippurate (PAH) transporter from winter flounder (Pseudopleuronectes americanus), designated fROAT, was cloned by functional expression in Xenopus laevis oocytes. fROAT is approximately 2.8 kbp in length and encodes a protein of 562 amino acids, related to the rat renal organic anion transporter ROAT1/OAT1 and the organic cation transporters OCT1 and OCT2. In oocytes, fROAT mediated probenecid-sensitive PAH uptake, with a K m for PAH of about 20 jiM, and inhibited by external glutarate (GA) (1 mM). The functional characteristics suggest that fROAT is the basolateral PAH/dicarboxylate exchanger of the flounder kidney.

Expression cloning in Xenopus laevis oocytes was used to isolate an organic anion transport protein from rat kidney. A cDNA library was constructed from sizefractionated poly(A) ؉ RNA and screened for probenecidsensitive transport of p-aminohippurate (PAH). A 2,227-base pair cDNA clone containing a 1,656-base pair open reading frame coding for a peptide 551 amino acids long was isolated and named ROAT1. ROAT1-mediated transport of 50 M [ 3 H]PAH was independent of imposed changes in membrane potential. Transport was significantly inhibited at 4°C, or upon incubation with other organic anions, but not by the organic cation tetraethylammonium, by the multidrug resistance ATPase inhibitor cyclosporin A, or by urate. External glutarate and ␣-ketoglutarate (1 mM), both counterions for basolateral PAH exchange, also inhibited transport, suggesting that ROAT1 is functionally similar to the basolateral PAH carrier. Consistent with this conclusion, PAH uptake was trans-stimulated in oocytes preloaded with glutarate, whereas the dicarboxylate methylsuccinate, which is not accepted by the basolateral exchanger, did not trans-stimulate. Finally, ROAT1-mediated PAH transport was saturable, with an estimated K m of 70 M. Each of these properties is identical to those previously described for the basolateral ␣-ketoglutarate/PAH exchanger in isolated membrane vesicles or intact renal tubules.Renal organic anion transport has been widely studied for more than a century, both as a prototypic transport process and as a primary means for removal of xenobiotics from the body. Because many foreign chemicals, including plant and animal toxins, drugs, and pesticides, are organic anions or are metabolized to organic anions, the renal organic anion secretory system plays a critical role in limiting or preventing their toxicity. Over the last decade, a great deal of progress has been made toward understanding the physiology of this system, particularly its coupling to metabolic energy. Thus, it is now well established that organic anion secretion is a complex process involving distinctly different proteins at the apical and basolateral membranes of the proximal tubule (Fig.

Chronic cadmium (Cd 2ϩ ) exposure results in renal proximal tubular cell damage. Delivery of Cd 2ϩ to the kidney occurs mainly as complexes with metallothionein-1 (molecular mass ϳ 7 kDa), freely filtered at the glomerulus. For Cd 2ϩ to gain access to the proximal tubule cells, these complexes are thought to be internalized via receptors for small protein ligands, such as megalin and cubilin, followed by release of Cd 2ϩ from metallothionein-1 in endosomal/lysosomal compartments. To investigate the role of megalin in renal cadmiummetallothionein-1 reabsorption, megalin expression and dependence of cadmium-metallothionein-1 internalization and cytotoxicity on megalin were studied in a renal proximal tubular cell model (WKPT-0293 Cl.2 cells). Expression of megalin was detected by reverse transcriptase-polymerase chain reaction and visualized by immunofluorescence both at the cell surface (live staining) and intracellularly (permeabilized cells). Internalization of Alexa Fluor 488-coupled metallothionein-1 was concentration-dependent, saturating at approximately 15 M. At 14.3 M, metallothionein-1 uptake could be significantly attenuated by 30.9 Ϯ 6.6% (n ϭ 4) by 1 M of the receptorassociated protein (RAP) used as a competitive inhibitor of cadmium-metallothionein-1 binding to megalin and cubilin. Consistently, cytotoxicity of a 24-h treatment with 7.14 M cadmium-metallothionein-1 was significantly reduced by 41.0 Ϯ 7.6%, 61.6 Ϯ 3.4%, and 26.2 Ϯ 1.8% (n ϭ 4 -5 each) by the presence of 1 M RAP, 400 g/ml anti-megalin antibody, or 5 M of the cubilin-specific ligand, apo-transferrin, respectively. Cubilin expression in proximal tubule cells was also confirmed at the mRNA and protein level. The data indicate that renal proximal tubular cadmium-metallothionein-1 uptake and cell death are mediated at least in part by megalin.

The kidney has recently emerged as an organ with a significant role in systemic iron (Fe) homeostasis. Substantial amounts of Fe are filtered by the kidney, which have to be reabsorbed to prevent Fe deficiency. Accordingly Fe transporters and receptors for protein-bound Fe are expressed in the nephron that may also function as entry pathways for toxic metals, such as cadmium (Cd), by way of "ionic and molecular mimicry". Similarities, but also differences in handling of Cd by these transport routes offer rationales for the propensity of the kidney to develop Cd toxicity. This critical review provides a comprehensive update on Fe transport by the kidney and its relevance for physiology and Cd nephrotoxicity. Based on quantitative considerations, we have also estimated the in vivo relevance of the described transport pathways for physiology and toxicology. Under physiological conditions all segments of the kidney tubules are likely to utilize Fe for cellular Fe requiring processes for metabolic purposes and also to contribute to reabsorption of free and bound forms of Fe into the circulation. But Cd entering tubule cells disrupts metabolic pathways and is unable to exit. Furthermore, our quantitative analyses contest established models linking chronic Cd nephrotoxicity to proximal tubular uptake of metallothionein-bound Cd. Hence, Fe transport by the kidney may be beneficial by preventing losses from the body. But increased uptake of Fe or Cd that cannot exit tubule cells may lead to kidney injury, and Fe deficiency may facilitate renal Cd uptake.