Kristen K. Evans

Transepithelial water and urea permeabilities of isolated perfused Munich-Wistar rat inner medullary thin limbs of Henle's loop

To better understand the role that water and urea fluxes play in the urine concentrating mechanism, we determined transepithelial osmotic water permeability (Pf) and urea permeability (Purea) in isolated perfused Munich-Wistar rat long-loop descending thin limbs (DTLs) and ascending thin limbs (ATLs). Thin limbs were isolated either from 0.5 to 2.5 mm below the outer medulla (upper inner medulla) or from the terminal 2.5 mm of the inner medulla. Segment types were characterized on the basis of structural features and gene expression levels of the water channel aquaporin 1, which was high in the upper DTL (DTLupper), absent in the lower DTL (DTLlower), and absent in ATLs, and the Cl-(1) channel ClCK1, which was absent in DTLs and high in ATLs. DTLupper Pf was high (3,204.5 ± 450.3 μm/s), whereas DTLlower showed very little or no osmotic Pf (207.8 ± 241.3 μm/s). Munich-Wistar rat ATLs have previously been shown to exhibit no Pf. DTLupper Purea was 40.0 ± 7.3 × 10(-5) cm/s and much higher in DTLlower (203.8 ± 30.3 × 10(-5) cm/s), upper ATL (203.8 ± 35.7 × 10(-5) cm/s), and lower ATL (265.1 ± 49.8 × 10(-5) cm/s). Phloretin (0.25 mM) did not reduce DTLupper Purea, suggesting that Purea is not due to urea transporter UT-A2, which is expressed in short-loop DTLs and short portions of some inner medullary DTLs close to the outer medulla. In summary, Purea is similar in all segments having no osmotic Pf but is significantly lower in DTLupper, a segment having high osmotic Pf. These data are inconsistent with the passive mechanism as originally proposed.

Basolateral choline transport in isolated rabbit renal proximal tubules.

Abstract Choline can undergo both net secretion and net reabsorption by renal proximal tubules, but at physiological plasma levels net reabsorption occurs. During this process, choline enters the cells at the luminal side down an electrochemical gradient via a specific transporter with a high affinity for choline. It appeared likely that choline was then transported out of the cells against an electrochemical gradient at the basolateral membrane by countertransport for another organic cation. This possibility was examined by studying net transepithelial reabsorption and basolateral uptake and efflux of [14C]choline in isolated S2 segments of rabbit renal proximal tubules. Basolateral uptake, which was inhibited by other organic cations such as tetraethylammonium (TEA), appeared to occur by the standard organic cation transport pathway. However, the addition of TEA to the bathing medium not only failed to trans-stimulate net transepithelial reabsorption and basolateral efflux of [14C]choline but it actually inhibited transepithelial reabsorption by @60%. The results do not support the presence of a countertransport step for choline against an electrochemical gradient at the basolateral membrane. Instead, they suggest that choline crosses this membrane by some form of carrier-mediated diffusion even during the reabsorptive process.

Basolateral Uptake of Nucleosides by Sertoli Cells Is Mediated Primarily by Equilibrative Nucleoside Transporter 1

The blood-testis barrier (BTB) prevents the entry of many xenobiotic compounds into seminiferous tubules thereby protecting developing germ cells. Understanding drug transport across the BTB may improve drug delivery into the testis. Members of one class of drug, nucleoside reverse transcriptase inhibitors (NRTIs), do penetrate the BTB, presumably through interaction with physiologic nucleoside transporters. By investigating the mechanism of nucleoside transport, it may be possible to design other drugs to bypass the BTB in a similar manner. We present a novel ex vivo technique to study transport at the BTB that employs isolated, intact seminiferous tubules. Using this system, we found that over 80% of total uptake by seminiferous tubules of the model nucleoside uridine could be inhibited by 100 nM nitrobenzylmercaptopurine riboside (NBMPR, 6-S-[(4-nitrophenyl)methyl]-6-thioinosine), a concentration that selectively inhibits equilibrative nucleoside transporter 1 (ENT1) activity. In primary cultured rat Sertoli cells, 100 nM NBMPR inhibited all transepithelial transport and basolateral uptake of uridine. Immunohistochemical staining showed ENT1 to be located on the basolateral membrane of human and rat Sertoli cells, whereas ENT2 was located on the apical membrane of Sertoli cells. Transepithelial transport of uridine by rat Sertoli cells was partially inhibited by the NRTIs zidovudine, didanosine, and tenofovir disoproxil fumarate, consistent with an interaction between these drugs and ENT transporters. These data indicate that ENT1 is the primary route for basolateral nucleoside uptake into Sertoli cells and a possible mechanism for nucleosides and nucleoside-based drugs to undergo transepithelial transport.

The multidrug transporter MATE1 sequesters OCs within an intracellular compartment that has no influence on OC secretion in renal proximal tubules.

Secretion of organic cations (OCs) across renal proximal tubules (RPTs) involves basolateral OC transporter (OCT)2-mediated uptake from the blood followed by apical multidrug and toxin extruder (MATE)1/2-mediated efflux into the tubule filtrate. Whereas OCT2 supports electrogenic OC uniport, MATE is an OC/H(+) exchanger. As assessed by epifluorescence microscopy, cultured Chinese hamster ovary (CHO) cells that stably expressed human MATE1 accumulated the fluorescent OC N,N,N-trimethyl-2-[methyl(7-nitrobenzo[c][l,2,5]oxadiazol-4-yl)amino]ethanaminium (NBD-MTMA) in the cytoplasm and in a smaller, punctate compartment; accumulation in human OCT2-expressing cells was largely restricted to the cytoplasm. A second intracellular compartment was also evident in the multicompartmental kinetics of efflux of the prototypic OC [(3)H]1-methyl-4-phenylpyridinium (MPP) from MATE1-expressing CHO cells. Punctate accumulation of NBD-MTMA was markedly reduced by coexposure of MATE1-expressing cells with 5 μM bafilomycin (BAF), an inhibitor of V-type H(+)-ATPase, and accumulation of [(3)H]MPP and [(3)H]NBD-MTMA was reduced by >30% by coexposure with 5 μM BAF. BAF had no effect on the initial rate of MATE1-mediated uptake of NBD-MTMA, suggesting that the influence of BAF was a secondary effect involving inhibition of V-type H(+)-ATPase. The accumulation of [(3)H]MPP by isolated single nonperfused rabbit RPTs was also reduced >30% by coexposure to 5 μM BAF, suggesting that the native expression in RPTs of MATE protein within endosomes can increase steady-state OC accumulation. However, the rate of [(3)H]MPP secretion by isolated single perfused rabbit RPTs was not affected by 5 μM BAF, suggesting that vesicles loaded with OCs(+) are not likely to recycle into the apical plasma membrane at a rate sufficient to provide a parallel pathway for OC secretion.

Body mass-specific Na+-K+-ATPase activity in the medullary thick ascending limb: implications for species-dependent urine concentrating mechanisms

In general, the mammalian whole body mass-specific metabolic rate correlates positively with maximal urine concentration (Umax) irrespective of whether or not the species have adapted to arid or mesic habitat. Accordingly, we hypothesized that the thick ascending limb (TAL) of a rodent with markedly higher whole body mass-specific metabolism than rat exhibits a substantially higher TAL metabolic rate as estimated by Na+-K+-ATPase activity and Na+-K+-ATPase α1-gene and protein expression. The kangaroo rat inner stripe of the outer medulla exhibits significantly higher mean Na+-K+-ATPase activity (~70%) compared with two rat strains (Sprague-Dawley and Munich-Wistar), extending prior studies showing rat activity exceeds rabbit. Furthermore, higher expression of Na+-K+-ATPase α1-protein (~4- to 6-fold) and mRNA (~13-fold) and higher TAL mitochondrial volume density (~20%) occur in the kangaroo rat compared with both rat strains. Rat TAL Na+-K+-ATPase α1-protein expression is relatively unaffected by body hydration status or, shown previously, by dietary Na+, arguing against confounding effects from two unavoidably dissimilar diets: grain-based diet without water (kangaroo rat) or grain-based diet with water (rat). We conclude that higher TAL Na+-K+-ATPase activity contributes to relationships between whole body mass-specific metabolic rate and high Umax. More vigorous TAL Na+-K+-ATPase activity in kangaroo rat than rat may contribute to its steeper Na+ and urea axial concentration gradients, adding support to a revised model of the urine concentrating mechanism, which hypothesizes a leading role for vigorous active transport of NaCl, rather than countercurrent multiplication, in generating the outer medullary axial osmotic gradient.

Isolation and perfusion of rat inner medullary vasa recta.

Outer medullary isolated descending vasa recta have proven to be experimentally tractable, and consequently much has been learned about outer medullary vasa recta endothelial transport, pericyte contractile mechanisms, and tubulovascular interactions. In contrast, inner medullary vasa recta have never been isolated from any species, and therefore isolated vasa recta function has never been subjected to in vitro quantitative evaluation. As we teased out inner medullary thin limbs of Henles loops from the Munich-Wistar rat, we found that vasa recta could be isolated using similar protocols. We isolated ∼30 inner medullary vasa recta from 23 adult male Munich-Wistar rats and prepared them for brightfield or electron microscopy, gene expression analysis by RT-PCR, or isolated tubule microperfusion. Morphological characteristics include branching and nonbranching segments exhibiting a thin endothelium, axial surface filaments radiating outward giving vessels a hairy appearance, and attached interstitial cells. Electron microscopy shows multiple cells, tight junctions, and either continuous or fenestrated endothelia. Isolated vasa recta express genes encoding the urea transporter UT-B and/or the fenestral protein PV-1, genes expressed in descending or ascending vasa recta, respectively. The transepithelial NaCl permeability (383.3 ± 60.0 × 10(-5) cm/s, mean ± SE, n = 4) was determined in isolated perfused vasa recta. Future quantitative analyses of isolated inner medullary vasa recta should provide structural and functional details important for more fully understanding fluid and solute flows through the inner medulla and their associated regulatory pathways.