Tonicity inversely modulates lipocalin-2 (Lcn2/24p3/NGAL) receptor (SLC22A17) and Lcn2 expression via Wnt/ß-catenin signaling in renal inner medullary collecting duct cells: implications for cell fate and bacterial infection.

BACKGROUND: We have previously evidenced apical expression of the 24p3/NGAL/lipocalin-2 receptor (Lcn2-R; SLC22A17) in inner medullary collecting duct (IMCD) cells, which are present in vivo in a hyperosmotic/-tonic environment that activates canonical Wnt/ß-catenin signaling. The localization of Lcn2-R in the inner medulla is intriguing considering local bacterial infections trigger toll-like receptor-4 (TLR-4)-mediated secretion of the bacteriostatic Fe3+-free (apo-)Lcn2. AIM: To determine the effects of osmolarity/tonicity changes, Wnt/ß-catenin and TLR-4 activation on Lcn2-R and Lcn2 expression and cell viability in rat primary IMCD and mouse (m)IMCD3 cells. METHODS: Normosmolarity/-tonicity was 300 mosmol/l whereas hyperosmolarity/-tonicity was induced by adding 100 mmol/l NaCl + 100 mmol/l urea (600 mosmol/l, 1-7 days). Lcn2-R and Lcn2 expression were determined by qPCR, immunoblotting, flow cytometry and immunofluorescence microscopy. ß-catenin was silenced by RNAi. Cell viability/death was determined with MTT and LDH release assays. TLR-4 was activated by bacterial lipopolysaccharides (LPS). RESULTS: Hyperosmotic/-tonic media upregulated Lcn2-R by ~4-fold and decreased Lcn2 expression/secretion, along with Wnt/ß-catenin activation, in IMCD cells. These effects of hyperosmotic/-tonic media on Lcn2-R/Lcn2 expression were reverted by normosmolarity/-tonicity, ß-catenin silencing and/or LPS. Exposure of cells with endogenous or stably overexpressing Lcn2-R to apo-Lcn2 or LPS decreased cell viability. CONCLUSIONS: Lcn2-R upregulation and Lcn2 downregulation via Wnt/ß-catenin may promote adaptive osmotolerant survival of IMCD cells in response to hyperosmolarity/-tonicity whereas Lcn2 upregulation and Lcn2-R downregulation via TLR-4 and/or normosmolarity/-tonicity may protect IMCD cells against bacterial infections and prevent autocrine death induction by Lcn2.

Molecular physiology of renal p-aminohippurate secretion.

Renal proximal tubules secrete various organic anions, including drugs and p-aminohippurate (PAH). Uptake of PAH from blood into tubule cells occurs by exchange with intracellular alpha-ketoglutarate and is mediated by the organic anion transporter 1. PAH exit into tubule lumen is species specific and may involve ATP-independent and -dependent transporters.

Genomic structure and in vivo expression of the human organic anion transporter 1 (hOAT1) gene.

The human organic anion transporter 1 (hOAT1) plays a key role in the secretion of an array of potentially toxic organic anions including many clinically important drugs. Here we report on the genomic cloning of hOAT1. A human genomic library was used for screening of a PAC (P1 artificial chromosome) clone applying PCR techniques. Sequencing of several restriction subclones and of a PCR-generated clone revealed that the hOAT1 gene spans 8.2 kb and is composed of 10 exons divided by 9 introns. RT-PCR studies in a human kidney specimen led to the detection of two new splice variants, hOAT1-3 and hOAT1-4, showing a 132-bp in-frame deletion. Using fluorescence in situ hybridization (FISH) we mapped the hOAT1 gene as a single signal to chromosome 11q13.1-q13.2. Additionally, 600 bp of the 5' flanking region was analyzed, illustrating the probable transcription start site at nt -280, a NF-kappaB-site at nt -397 and several putative transcription factor binding sites.

Structure of renal organic anion and cation transporters.

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.

Voltage-driven p-aminohippurate, chloride, and urate transport in porcine renal brush-border membrane vesicles.

p-Aminohippurate (PAH) and urate are secreted into the proximal tubule lumen across the brush-border membrane. Here we used brush-border membrane vesicles from pig kidney to study PAH and urate transport. Efflux and influx of [3H]PAH were influenced by K+-diffusion potentials indicating electrogenic PAH transport. An outside>inside PAH concentration difference accelerated voltage-sensitive, Na+-coupled D-glucose uptake as efficiently as did an outside>inside Cl- concentration difference, suggesting comparable conductances for PAH and Cl- in brush-border membrane vesicles. Up to 1 mM of the uricosurics indacrinone, tienilic acid, losartan and probenecid, as well as of the stilbenes, DIDS and SITS, and of the loop diuretics furosemide and bumetanide inhibited voltage-driven PAH uptake, but not, or only slightly, voltage-driven Cl- uptake. Voltage-driven [14C]urate uptake, however, was inhibited by 0.1 mM DIDS, 0.2 mM losartan and 0.5 mM probenecid to a similar extent as [3H]PAH uptake. One millimolar pyrazinoic acid, oxonate, xanthine and adenosine inhibited neither [3H]PAH nor [14C]urate uptake. These results suggest that PAH and urate share an anion conductance which is distinct from the Cl- conductance and is probably not the same as a recently identified urate channel (Leal-Pinto E et a]. J Biol Chem 272:617-625, 1997).

Expression cloning and characterization of a novel sodium-dicarboxylate cotransporter from winter flounder kidney.

A cDNA coding for a Na+-dicarboxylate cotransporter, fNaDC-3, from winter flounder (Pseudopleuronectes americanus) kidney was isolated by functional expression in Xenopus laevis oocytes. The fNaDC-3 cDNA is 2384 nucleotides long and encodes a protein of 601 amino acids with a calculated molecular mass of 66.4 kDa. Secondary structure analysis predicts at least eight membrane-spanning domains. Transport of succinate by fNaDC-3 was sodium-dependent, could be inhibited by lithium, and evoked an inward current. The apparent affinity constant (Km) of fNaDC-3 for succinate of 30 microM resembles that of Na+-dicarboxylate transport in the basolateral membrane of mammalian renal proximal tubules. The substrates specific for the basolateral transporter, 2,3-dimethylsuccinate and cis-aconitate, not only inhibited succinate uptake but also evoked inward currents, proving that they are transported by fNaDC-3. Succinate transport via fNaDC-3 decreased by lowering pH, as did citrate transport, although much more moderately. These characteristics suggest that fNaDC-3 is a new type of Na+-dicarboxylate transporter that most likely corresponds to the Na+-dicarboxylate cotransporter in the basolateral membrane of mammalian renal proximal tubules.

Expression cloning and characterization of ROAT1. The basolateral organic anion transporter in rat 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 size-fractionated poly(A)+ RNA and screened for probenecid-sensitive 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 mu M [3H]PAH was independent of imposed changes in membrane potential. Transport was significantly inhibited at 4 degrees 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 alpha-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 Km of 70 mu M. Each of these properties is identical to those previously described for the basolateral alpha-ketoglutarate/PAH exchanger in isolated membrane vesicles or intact renal tubules.

Expression cloning and characterization of a renal organic anion transporter from winter flounder.

The cDNA coding for a renal p-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 Km for PAH of about 20 microM, and inhibited by external glutarate (GA) (1 mM). The functional characteristics suggest that fROAT is the basolateral PAH/dicarboxylate exchanger of the flounder kidney.

Functional expression of renal organic anion transport in Xenopus laevis oocytes.

Secretion of organic anions by the kidney plays a critical role in the elimination of toxic agents from the body. Recent findings in isolated membranes and intact tissue have demonstrated the participation of multiple transport proteins in this process. As a first step toward molecular characterization of these proteins through expression cloning, the studies reported below demonstrate functional expression of both fumarate- and lithium-sensitive glutarate and probenecid-sensitive p-aminohippurate transport in Xenopus oocytes injected with rat kidney poly(A)+ RNA. Maximal increase in substrate uptake over buffer-injected controls was reached by 5 days after mRNA injection. Expression of size-fractionated mRNA indicated that the active species with respect to both transport activities were in the range of 1.8 to 3.5 kb.

Taurine transport by rabbit kidney brush-border membranes: coupling to sodium, chloride, and the membrane potential.

Ion dependence and electrogenicity of taurine uptake were studied in rabbit renal outer cortical brush-border membrane vesicles isolated by differential precipitation. Na+-D-glucose cotransport was followed in parallel to monitor changes in the membrane potential. Concentrative taurine flux was dependent on a chemical and/or an electrical Na+ gradient (K+ diffusion potential) and could be completely inhibited by other beta-amino acids. It displayed a specific anion requirement (Cl- greater than or equal to Br- much greater than SCN- greater than I- greater than NO-3). At chemical Na+ equilibrium, Cl- gradients, depending on their orientation, stimulated or inhibited taurine uptake more than could be attributed solely to electrical anion effects, although a Cl- gradient alone could not energize an overshoot. Furthermore, taurine tracer exchange was significantly stimulated by Cl- as well as Br-. The Cl- stoichiometry was found to be one, whereas taurine transport, in the presence of Cl-, was sigmoidally related to the Na+ concentration, resulting in a coupling ratio of 2 to 3 Na+: 1 taurine. Upon Cl- replacement with gluconate, taurine uptake showed a reduced potential sensitivity and was no longer detectably affected by the Na+ concentration (up to 150 mM). These results suggest a 2 to 3 Na+ :1 Cl- :1 taurine cotransport mechanism driven mainly by the Na+ gradient, which is sensitive to the membrane potential due to a negatively charged empty carrier. Cl- appears to stimulate taurine flux primarily by facilitating the formation of the translocated solute-carrier complex.

Renal handling of taurine, L-alanine, L-glutamate and D-glucose in Opsanus tau: studies on isolated brush border membrane vesicles.

Renal brush border membrane vesicles (bbmv) from the aglomerular toadfish (Opsanus tau), isolated by differential precipitation, were tested for their ability to actively translocate (i) taurine, known to be secreted by the kidney of several marine teleosts, and (ii) L-alanine, L-glutamic acid, and D-glucose, solutes that are normally reabsorbed in the filtering nephron. Vesicular taurine uptake displayed a Na+ dependence. Transport was greatest under conditions of an inward-directed Na+ gradient, but a significant stimulation by Na+ over K+ could also be observed in the absence of a salt gradient. At high extravesicular K+, the addition of valinomycin reduced taurine uptake. Na+-dependent 3H-taurine flux was almost completely inhibited by non-labeled taurine (tracer replacement) or beta-alanine, but was unaffected by L-alanine. Replacement of medium chloride by SCN- or NO3- in the presence of Na+ resulted in significantly lower uptake rates under both anion gradient and anion equilibrium conditions, whereas Br- could almost fully substitute for the stimulatory Cl- action. These results indicate the presence of an electrogenic Na+-cotransport mechanism with specificity for beta-amino acids in the toadfish renal brush border. Whether the system under physiological conditions mediates reabsorption or secretion of taurine remains to be determined. Toadfish bbmv also translocated L-alanine and L-glutamic acid in a Na+-dependent manner. Possible roles for these most likely reabsorptive transport systems in a non-filtering kidney are discussed. D-glucose uptake, however, appeared to occur via Na+-independent pathways, since it was not affected by phlorizin in the presence of Na+, or by Na+ replacement.

Ionic requirements of peritubular taurine transport in Fundulus kidney.

A peritubular mechanism for active taurine uptake, similar to that previously found in the flounder, was demonstrated in killifish (Fundulus heteroclitus) teased renal tubules. Other beta-amino acids and glycine inhibited taurine transport. Uptake exhibited a nearly absolute requirement for external Na+. The stoichiometric relationship between Na+ and taurine was found to be 2:1. Medium Cl- markedly stimulated the Na+-dependent taurine uptake, but none of the other small monovalent anions tested (NO3-, Br-, SCN-) could substitute for Cl- in activating taurine transport. Cl- replacement resulted in a significant decrease in V max, whereas the affinity of the carrier for taurine appeared to be unaffected. In the absence of Cl- the Na+ dependence of taurine influx was reduced to a stoichiometric relationship of 1.4 Na+:1 taurine, indicating an effect of Cl- on the binding of Na+ to the transport system. A model for Na+ -Cl- -taurine cotransport is presented.