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Hypertonicity increases urea transport independently of, as well as synergistically with, vasopressin in the inner medullary collect duct (IMCD). We previously showed that hypertonicity does not increase the level of cAMP in the IMCD, but it does increase the level of intracellular calcium. Since we also showed that hypertonicity increases both the phosphorylation and biotinylation of the urea transporters UT-A1 and UT-A3, this would suggest involvement of a calcium-dependent protein kinase in the regulation of urea transport in the inner medulla. In this study, we investigated whether protein kinase C (PKC), which is present in the IMCD, is a regulator of urea permeability. We tested the effect of PKC inhibitors and activators on urea permeability in the isolated, perfused rat terminal IMCD. Increasing osmolality from 290 to 690 mosmol/kg[H.sub.2]O significantly stimulated (doubled) urea permeability; it returned to control levels on inhibition of PKC with either 10 [micro]M chelerythrine or 50 [micro]M rottlerin. To determine the potential synergy between vasopressin and PKC, phorbol dibutyrate (PDBu) was used to stimulate PKC. Vasopressin stimulated urea permeability 247%. Although PDBu alone did not change basal urea permeability, in the presence of vasopressin, it significantly increased urea permeability an additional 92%. The vasopressin and PDBu-stimulated urea permeability was reduced to AVP alone levels by inhibition of PKC. We conclude that hypertonicity stimulates urea transport through a PKC-mediated phosphorylation. Whether PKC directly phosphorylates UT-A1 and/or UT-A3 or phosphorylates it as a consequence of a cascade of activations remains to be determined. tubule perfusion; calcium signaling; urine concentration; vasopressin; hypertonicity doi: 10.1152/ajprenal.00322.2010

Dynamin is a large GTPase involved in several distinct modes of cell endocytosis. In this study, we examined the possible role of dynamin in UT-A1 internalization. The direct relationship of UT-A1 and dynamin was identified by coimmunoprecipitation. UT-A1 has cytosolic [NH.sub.2] and COOH termini and a large intracellular loop. Dynamin specifically binds to the intracellular loop of UT-A1, but not the [NH.sub.2] and COOH termini. In cell surface biotinylation experiments, coexpression of dynamin and UT-A l in HEK293 cells resulted in a decrease of UT-A 1 cell surface expression. Conversely, cells expressing dynamin mutant K44A, which is deficient in GTP binding, showed an increased accumulation of UT-A1 protein on the cell surface. Cell plasma membrane lipid raft fractionation experiments revealed that blocking endocytosis with dynamin K44A causes UT-A1 protein accumulation in both the lipid raft and nonlipid raft pools, suggesting that both caveolae--and clathrin-mediated mechanisms may be involved in the internalization of UT-A1. This was further supported by 1) small interfering RNA to knock down either caveolin-I or [mu]2 reduced UT-A1 internalization in HEK293 cells and 2) inhibition of either the caveolae pathway by methyl-[beta]-cyclodextrin or the clathrin pathway by concanavalin A caused UT-A1 cell membrane accumulation. Functionally, overexpression of dynamin, caveolin, or [mu]2 decreased UT-A1 urea transport activity and decreased UT-A1 cell surface expression. We conclude that UT-A1 endocytosis is dynamin-dependent and mediated by both caveolae--and clathrin-coated pit pathways. membrane protein; urea transport; endocytosis doi: 10.1152/ajprenal.00718.2009

Of the three major protein variants produced by the UT-A gene (UT-A1, UT-A2, and UT-A3) UT-A1 is the largest. It contains UT-A3 as its N[H.sub.2]-terminal half and UT-A2 as its COOH-terminal half. When being part of UT-A1, UT-A3 and UT-A2 are joined by a segment, Lp, whose central part, Lc, is not part of UT-A3 or UT-A2 but is present only in UT-A1. Lc contains the phosphorylation sites S486 and S499 that are involved in protein kinase A-dependent activation, as well as the binding site for snapin, a protein involved in soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor (SNARE)-mediated vesicle trafficking and fusion to the plasma membrane. We attached Lc to UT-A2 and UT-A3 to test how these phosphorylation sites influenced their urea transport activity. Adding Lc to UT-A2 conferred stimulation by cAMP to the cAMP-unresponsive UT-A2, and adding Lc to UT-A3 did not further enhance its already existing cAMP response. These findings suggest that the responsiveness to vasopressin that is observed with UT-A1 can be introduced into the unresponsive UT-A2 variant through the Lc segment that is unique to UT-A1. In UT-A3, however, the Lc segment plays no significant role in its activation by cAMP. In addition, the Lc segment also gave UT-A2 the ability to bind snapin and, in Xenopus oocytes, to be stimulated in its urea transport activity by snapin and syntaxins 3 and 4, in the same way as UT-A1. phosphorylation site; protein kinase A-dependent activation doi: 10.1152/ajpcell.00497.2009.

The sodium-bicarbonate cotransporter NBCn1 (SLC4A7) is an acid-base transporter that normally moves [Na.sup.+] and HC[O.sup.-.sub.3] into the cell. This membrane protein is sensitive to cellular and systemic pH changes. We examined NBCn1 expression and localization in the brain and its response to chronic metabolic acidosis. Two new NBCn1 antibodies were generated by immunizing a rabbit and a guinea pig. The antibodies stained neurons in a variety of rat brain regions, including hippocampal pyramidal neurons, dentate gyrus granular neurons, posterior cortical neurons, and cerebellar Purkinje neurons. Choroid plexus epithelia were also stained. Double immunofluorescence labeling showed that NBCn1 and the postsynaptic density protein PSD-95 were found in the same hippocampal CA3 neurons and partially colocalized in dendrites. PSD-95 was pulled down from rat brain lysates with the GST/NBCn1 fusion protein and was also coimmunoprecipitated with NBCn1. Chronic metabolic acidosis was induced by feeding rats with normal chow or 0.4 M HCl-containing chow for 7 days. Real-time PCR and immunoblot showed upregulation of NBCn1 mRNA and protein in the hippocampus of acidotic rats. NBCn1 immunostaining was enhanced in CA3 neurons, posterior cortical neurons, and cerebellar granular cells. Intraperitoneal administration of N-methyl-D-aspartate caused neuronal death determined by caspase-3 activity, and this effect was more severe in acidotic rats. Administering N-methyl-D-aspartate also inhibited NBCn1 upregulation in acidotic rats. We conclude that NBCn1 in neurons is upregulated by chronic acid loads, and this upregulation is associated with glutamate excitotoxicity. acid/base; pH; ion transporter doi: 10.1152/ajpcell.00492.2009.

Vasopressin is the primary hormone regulating urine-concentrating ability. Vasopressin phosphorylates the UT-Al urea transporter in rat inner medullary collecting ducts (IMCDs). To assess the effect of UT-Al phosphorylation at $486, we developed a phospho-specific antibody to S486-UT-A1 using an 11 amino acid peptide antigen starting from amino acid 482 that bracketed $486 in roughly the center of the sequence. We also developed two stably transfected mlMCD3 cell lines: one expressing wild-type UT-Al and one expressing a mutated form of UT-Al, S486A/S499A, that is unresponsive to protein kinase A. Forskolin stimulates urea flux in the wild-type UT-Al-mIMCD3 cells but not in the S486A/S499A-UT-Al-mIMCD3 cells. The phospbo-S486-UT-Al antibody identified UT-Al protein in the wild-type UT-Al-mIMCD3 cells but not in the S486A/S499A-UT-Al-mIMCD3 cells. In rat IMCDs, forskolin increased the abundance of phospbo-S486-UT-Al (measured using the phospho-S486 antibody) and of total UT-Al phosphorylation (measured by 32p incorporation). Forskolin also increased the plasma membrane accumulation of phospho-S486-UT-Al in rat IMCD suspensions, as measured by biotinylation. In rats treated with vasopressin in vivo, the majority of the phospho-S486-UT-Al appears in the apical plasma membrane. In summary, we developed stably transfected mIMCD3 cell lines expressing UT-Al and an S486-UT-Al phospho-specific antibody. We confirmed that vasopressin increases UT-Al accumulation in the apical plasma membrane and showed that vasopressin phosphorylates UT-Al at $486 in rat IMCDs and that the S486-phospho-UT-Al form is primarily detected in the apical plasma membrane. urea transporter; phospho-specific; IMCD3; vasopressin; forskolin doi: 10.1152/ajprenal.00682.2009.

Patients receiving lithium therapy, an effective treatment for bipolar disorder, often present with acquired nephrogenic diabetes insipidus. The nephrotoxic effects of lithium can be detected 3 wk after the start of treatment and many of these symptoms may disappear in a few weeks after lithium use is stopped. Most patients, however, still have a urine-concentrating defect years after ending treatment. This prompted an investigation of the transporters involved in the urine concentration mechanism, UT-A1, UT-A3, aquaporin-2 (AQP2), and NKCC2, after discontinuing lithium therapy. Sprague-Dawley rats fed a [Li.sub.2]C[O.sub.3]-supplemented diet produced large volumes of dilute urine after 14 days. After lithium treatment was discontinued, urine osmolality returned to normal within 14 days but urine volume and urine urea failed to reach basal levels. Western blot and immunohistochemical analyses revealed that both urea transporters UT-A1 and UT-A3 were reduced at 7 and 14 days of lithium treatment and both transporters recovered to basal levels 14 days after discontinuing lithium administration. Similar analyses demonstrated a decrease in AQP2 expression after 7 and 14 days of lithium therapy. AQP2 expression increased over the 7 and 14 days following the cessation of lithium but failed to recover to normal levels. NKCC2 expression was unaltered during the 14-day lithium regimen but did increase 14 days after the treatment was stopped. In summary, the rapid restoration of UT-A1 and UT-A3 as well as the increased expression of NKCC2 are critical components to the reestablishment of urine concentration after lithium treatment. urea transporter; aquaporin-2; acquired nephrogenic diabetes insipidus doi:10.1152/ajprenal.00424.2009

Mistry AC, Mallick R, Klein JD, Weimbs T, Sands JM, Frohlich O. Syntaxin specificity of aquaporins in the inner medullary collecting duct. Am J Physiol Renal Physiol 297: F292-F300, 2009. First published June 10, 2009; doi:10.1152/ajprenal.00196.2009.-Proper targeting of the aquaporin-2 (AQP2) water channel to the collecting duct apical plasma membrane is critical for the urine concentrating mechanism and body water homeostasis. However, the trafficking mechanisms that recruit AQP2 to the plasma membrane are still unclear. Snapin is emerging as an important mediator in the initial interaction of trafficked proteins with target soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptor (tSNARE) proteins, and this interaction is functionally important for AQP2 regulation. We show that in AQP2-Madin-Darby canine kidney ceils subjected to adenoviral-mediated expression of both snapin and syntaxins, the association of AQP2 with both syntaxin-3 and syntaxin-4 is highly enhanced by the presence of snapin. In pull-down studies, snapin detected AQP2, syntaxin-3, syntaxin-4, and SNAP23 from the inner medullary collecting duct. AQP2 transport activity, as probed by AQP2's urea permeability, was greatly enhanced in oocytes that were coinjected with cRNAs of SNARE components (snapin+syntaxin-3+SNAP23) over those injected with AQP2 cRNA alone. It was not enhanced when syntaxin-3 was replaced by syntaxin-4 (snapin+syntaxin4+SNAP23). On the other hand, the latter combination significantly enhanced the transport activity of the related AQP3 water channel while the presence of syntaxin-3 did not. This AQP-syntaxin interaction agrees with the polarity of these proteins' expression in the inner medullary collecting duct epithelium. Thus our findings suggest a selectivity of interactions between different aquaporin and syntaxin isoforms, and thus in the regulation of AQP2 and AQP3 activities in the plasma membrane. Snapin plays an important role as a linker between the water channel and the t-SNARE complex, leading to the fusion event, and the pairing with specific t-SNAREs is essential for the specificity of membrane recognition and fusion. urea transport; UT-A1; lithium; cAMP; MDCK cells

The cell plasma membrane contains specialized microdomains called lipid rafts which contain high amounts of sphingolipids and cholesterol. Lipid rafts are involved in a number of membrane protein functions. The urea transporter UT-A1, located in the kidney inner medullary collecting duct (IMCD), is important for urine concentrating ability. In this study, we investigated the possible role of lipid rafts in UT-A1 membrane regulation. Using sucrose gradient cell fractionation, we demonstrated that UT-A1 is concentrated in the caveolaerich fraction both in stably expressing UT-A1 HEK293 cells and in freshly isolated kidney IMCD suspensions. In these gradients, UT-A1 at the cell plasma membrane is codistributed with caveolin-1, a major component of caveolae. The colocalization of UT-A1 in lipid rafts/ caveolae was further confirmed in isolated caveolae from UT-A1-HEK293 cells. The direct association of UT-A1 and caveolin-1 was identified by immunoprecipitation and GST pull-down assay. Examination of internalized UT-A1 in pEGFP-UT-A1 transfected HEK293 cells fluorescent overlap with labeled cholera toxin subunit B, a marker of the caveolae-mediated endocytosis pathway. Disruption of lipid rafts by methyl-[beta]-cyclodextrin or knocking down caveolin-1 by small-interference RNA resulted in UT-A1 cell membrane accumulation. Functionally, overexpression of caveolin-1 in oocytes decreased UT-A1 urea transport activity and UT-A1 cell surface expression. Our results indicate that lipid rafts/caveolae participate in UT-A1 membrane regulation and this effect is mediated via a direct interaction of caveolin-1 with UT-A1. membrane protein; inner medulla; endocytosis; urea transport

In our previous studies of varying osmotic diuresis, UT-A1 urea transporter increased when urine and inner medullary (IM) interstitial urea concentration decreased. The purposes of this study were to examine 1) whether IM interstitial tonicity changes with different urine urea concentrations during osmotic dieresis and 2) whether the same result occurs even if the total urinary solute is decreased. Rats were fed a 4% high-salt diet (HSD) or a 5% high-urea diet (HUD) for 2 wk and compared with the control rats fed a regular diet containing 1% NaCl. The urine urea concentration decreased in HSD but increased in HUD. In the IM, UT-A1 and UT-A3 urea transporters, CLC-K1 chloride channel, and tonicity-enhanced binding protein (TonEBP) transcription factor were all increased in HSD and decreased in HUD. Next, rats were fed an 8% low-protein diet (LPD) or a 0.4% low-salt diet (LSD) to decrease the total urinary solute. Urine urea concentration significantly decreased in LPD but significantly increased in LSD. Rats fed the LPD had increased UT-A1 and UT-A3 in the IM base but decreased in the IM tip, resulting in impaired urine concentrating ability. The LSD rats had decreased UT-A1 and UT-A3 in both portions of the IM. CLC-K1 and TonEBP were unchanged by LPD or LSD. We conclude that changes in CLC-K1, UT-A1, UT-A3, and TonEBP play important roles in the renal response to osmotic diuresis in an attempt to minimize changes in plasma osmolality and maintain water homeostasis. UT-A3 urea transporter; CLC-K1 chloride channel; TonEBP transcription factor

Osmotic reabsorption of water through aquaporin-2 (AQP2) in the inner medulla is largely dependent on the urea concentration gradients generated by urea transporter (UT) isoforms. Vasopressin (AVP) increases expression of both AQP2 and UT-A isoforms. Activation of the P2Y2 receptor (P2Y2-R) in the medullary collecting duct inhibits AVP-induced water flow. To gain further insights into the overarching effect of purinergic signaling on urinary concentration, we compared the protein abundances of AQP2 and UT-A isoforms between P2Y2-R knockout (KO) and wild-type (WT) mice under basal conditions and following AVP administration. Under basal conditions (a gel diet for 10 days), KO mice concentrated urine to a significantly higher degree, with 1.8-, 1.66-, and 1.29-fold higher protein abundances of AQP2, UT-A1, and UT-A2, respectively, compared with WT, despite comparable circulating AVP levels in both groups. Infusion of 1-desamino-8-D-arginine vasopressin (dDAVP; desmopressin; 1 ng/h sc) for 5 days resulted in 2.14-, 2.6-, and 2.22-fold higher protein abundances of AQP2, AQP3, and UT-A1, respectively, in the inner medullas of KO mice compared with WT mice. In response to acute (45 min) stimulation by AVP (0.2 unit/mouse sc), UT-A1 protein increased by 1.39- and 1.54-fold in WT and KO mice, respectively. These data suggest that genetic deletion of P2Y2-R results in increased abundances of key proteins involved in urinary concentration in the inner medulla, both under basal conditions and following AVP administration. Thus purinergic regulation may play a potential overarching role in balancing the effect of AVP on the urinary concentration mechanism. collecting duct; arginine vasopressin; aquaporin; urea transporters; extracellular nucleotides

UT-A1 is the primary urea transporter in the apical plasma membrane responsible for urea reabsorption in the inner medullary collecting duct. Although the physiological function of UT-A1 has been well established, the molecular mechanisms that regulate its activity are less well understood. Analysis of the UT-A1 amino acid sequence revealed a potential MDM2 E3 ubiquitin ligase-binding motif in the large intracellular loop of UTA1, suggesting that UT-A1 urea transporter protein may be regulated by the ubiquitin-proteasome pathway. Here, we report that UT-A1 is ubiquitinated and degraded by the proteasome but not the lysosome proteolytic pathway. Inhibition of proteasome activity causes UT-A1 cell surface accumulation and concomitantly increases urea transport activity. UT-A1 interacts directly with MDM2; the binding site is located in the NH2-terminal p53-binding region of MDM2. MDM2 mediates UT-A1 ubiquitination both in vivo and in vitro. Overexpression of MDM2 promotes UT-A1 degradation. The mechanism is likely to be physiologically important as UT-A1 ubiquitination was identified in kidney inner medullary tissue. The ubiquitin-proteasome degradation pathway provides an important novel mechanism for UT-A1 regulation. proteolysis; membrane protein; urea transport; trafficking

The UT-A 1 and UT-A3 urea transporters are expressed in the terminal inner medullary collecting duct (IMCD) and play an important role in the production of concentrated urine. We showed that both hyperosmolarity and vasopressin increase urea permeability in perfused rat terminal IMCDs and that UT-A1 and UT-A3 accumulate in the plasma membrane in response to vasopressin. In this study, we investigated whether hyperosmolarity causes UT-A1 and/or UT-A3 to accumulate in the plasma membrane or represents a complimentary stimulatory pathway. Rat IMCD suspensions were incubated in 450 vs. 900 mosM solutions. We biotinylated the IMCD surface proteins, collected, and analyzed them. Membrane accumulation was assessed by Western blotting of the biotinylated protein pool probed with anti-UT-A1 or anti-UT-A3. We studied the effect of NaCl, urea, and sucrose as osmotic agents. Membrane-associated UT-A1 and UT-A3 increased relative to control levels when either NaCl (UT-A1 increased 37 [+ or -] 6%; UT-A3 increased 46 [+ or -] 13%) or sucrose (UT-A1 increased 81 [+ or -] 13%; UT-A3 increased 60 [+ or -] 8%) was used to increase osmolarity. There was no increase in membrane UT-A1 or UT-A3 when urea was added. Analogously, UT-A1 phosphorylation was increased in NaCl- and sucrose- but not in urea-based hyperosmolar solutions. Hypertonicity also increased UT-A3 phosphorylation. We conclude that the increase in the urea permeability in response to hyperosmolarity reflects both UT-A1 and UT-A3 movement to the plasma membrane and may be a direct response to tonicity. Furthermore, this movement is accompanied by, and may require, increased phosphorylation in response to hypertonicity. renal; osmolality; concentrating mechanism; trafficking

The UT-A1 urea transporter plays an important role in the urine concentrating mechanism. Vasopressin (or cAMP) increases urea permeability in perfused terminal inner medullary collecting ducts and increases the abundance of phosphorylated UT-A1, suggesting regulation by phosphorylation. We performed a phosphopeptide analysis that strongly suggested that a PKA consensus site(s) in the central loop region of UT-A1 was/were phosphorylated. Serine 486 was most strongly identified, with other potential sites at serine 499 and threonine 524. Phosphomutation constructs of each residue were made and transiently transfected into LLC-PK1 cells to assay for UT-A1 phosphorylation. The basal level of UT-A1 phosphorylation was unaltered by mutation of these sites. We injected oocytes, assayed [14C]urea flux, and determined that mutation of these sites did not alter basal urea transport activity. Next, we tested the effect of stimulating cAMP production with forskolin. Forskolin increased wild-type UT-A1 and T524A phosphorylation in LLC-PKI cells and increased urea flux in oocytes. In contrast, the $486A and $499A mutants demonstrated loss of forskolin-stimulated UT-A 1 phosphorylation and reduced urea flux. In LLC-[PK.sup.1] cells, we assessed biotinylated UT-A1. Wild-type UTA1, $486A, and $499A accumulated in the membrane in response to forskolin. However, in the S486A/S499A double mutant, forskolinstimulated UT-A1 membrane accumulation and urea flux were totally blocked. We conclude that the phosphorylation of UT-A1 on both serines 486 and 499 is important for activity and that this phosphorylation may be involved in UT-A1 membrane accumulation. protein kinase A consensus sites; protein kinase A; phosphorylation mutation; membrane trafficking; forskolin

Volume depletion due to persistent glucosuria-induced osmotic diuresis is a significant problem in uncontrolled diabetes mellitus (DM). Angiotensin II receptor blockers (ARBs), such as candesartan, slow the progression of chronic kidney disease in patients with DM. However, mice with genetic knockout of components of the renin-angiotensin system have urine concentrating defects, suggesting that ARBs may exacerbate the volume depletion. Therefore, the effect of candesartan on UT-A1, UT-A3, NKCC2, and aquaporin-2 (AQP2) protein abundances was determined in control and 3-wk DM rats. Aldosterone levels in control rats (0.36 [+ or -] 0.06 nM) and candesartan-treated rats (0.34 [+ or -] 0.14 nM) were the same. DM rats had higher aldosterone levels (1.48 [+ or -] 0.37 nM) that were decreased by candesartan (0.97 [+ or -] 0.26 nM). Western analysis showed that UT-A1 expression was increased in DM rats compared with controls in inner medullary (IM) tip (158 [+ or -] 13%) and base (120 [+ or -] 25%). UT-A3 abundance was increased in IM tip (123 [+ or -] 11%) and base (146 [+ or -] 17%) of DM rats vs. controls. UT-A3 was unchanged in candesartan-treated control rats. In candesartan-treated DM rats, UT-A3 increased in IM tip (160 [+ or -] 14%) and base (210 [+ or -] 19%). Candesartan-treated DM rats had slightly higher AQP2 in IM (46%, P < 0.05) vs. control rats. NKCC2/BSC1 was increased 145 [+ or -] 10% in outer medulla of DM vs. control rats. We conclude that candesartan augments compensatory changes in medullary transport proteins, reducing the losses of solute and water during uncontrolled DM. These changes may represent a previously unrecognized beneficial effect of type 1 ARBs in DM. angiotensin II; UT-A urea transporter; aquaporin; AQP2; NKCC2

In lithium-induced nephrogenic diabetes insipidus (NDI), alterations in renal medullary osmolyte concentrations have been assumed but never investigated. Amiloride can modify lithium-induced NDI, but the impact of amiloride in lithium-induced NDI on renal medullary osmolytes, aquaporins, and urea transporters is unknown and is the basis of this study. Rats fed lithium (60 mmol/kg dry food) over 4 wk developed NDI. Urine osmolality fell to 287 [+ or -] 19 mosmol/kg[H.sub.2]O (controls 1,211 [+ or -] 90 mosmol/kg[H.sub.2]O). Organic osmolytes in the renal medulla showed significant decreases compared with controls [inositol 221 [+ or -] 35 to 85 [+ or -] 10 mmol/kg protein; sorbitol 35 [+ or -] 9 to 3 [+ or -] 1 mmol/kg protein; glycerophosphorylcholine (GPC) 352 [+ or -] 80 to 91 [+ or -] 20 mmol/kg protein; and glycine betaine 69 [+ or -] 11 to 38 [+ or -] 38 mmol/kg protein]. Medullary urea content fell from 2,868 [+ or -] 624 to 480 [+ or -] 117 mmol/kg protein. Concurrent administration of amiloride (0.2 mmol/1) in the drinking water restored urine osmolality (1,132 [+ or -] 154 mosmol/ kg[H.sub.2]O), and reduced urine volume. Medullary osmolyte content were restored to control values (inositol, 232 [+ or -] 12; sorbitol 32 [+ or -] 6; GPC, 244 [+ or -] 26; glycine betaine, 84 [+ or -] 5 mmol/kg protein). Medullary urea rose to 2,122 [+ or -] 305 mmol/kg protein. Reduced AQP2, AQP3, and urea transporter (UT-A1) expression was significantly reversed following amiloride therapy. Data presented here provide further understanding of how amiloride may substantially restore the lithium-induced impaired renal concentrating mechanism. aquaporin; osmolytes; urea transporter

Trans-epithelial tracer urea flux across Madin-Darby canine kidney (MDCK) cells permanently expressing the urea transporter UT-Al is stimulated by agents that activate the cAMP signaling pathway, such as vasopressin or forskolin, thus mimicking the activation of urea permeability in the inner medullary collecting duct in the presence of vasopressin. Here, we report that UT-A1-mediated urea flux is also activated two-to-threefold over background by exposing the cells to media containing LiCl. This is in contrast to reports on cortical and medullary collecting duct tubules where acute and chronic exposure to lithium (Li) suppresses the osmotic water permeability, which is also regulated by cAMP levels. The Li concentration dependence of urea flux activation was linear up to 150 mM Li. Li activated only from the basolateral side where its effect was inhibited by amiloride, presumably because Li entered the cells through a basolateral Na-H exchanger. Li and IBMX, which also weakly activated urea flux, greatly augmented each others' stimulatory effect on urea flux. However, cellular cAMP levels did not rise commensurately with urea fluxes, and even though Li augments the activation by forskolin, it greatly inhibits the forskolin-induced formation of cAMP. These results suggest that the effect of Li in this MDCK model of renal cells does not involve cAMP or at least utilizes an additional signaling pathway independent of cAMP. cyclic AMP; Madin-Darby canine kidney cells

UT-A1 is regulated by vasopressin and is localized to the apical membrane and intracellular compartment of inner medullary collecting duct (IMCD) cells. UT-A3 is also expressed in the IMCD and is regulated by forskolin in heterologous systems. The goal of the present study is to investigate mechanisms by which vasopressin regulates UT-A3 in rat IMCD. In fresh suspensions of rat IMCD, forskolin increases the phosphorylation of UT-A3, similar to UT-A1. Biotinylation studies indicate that UT-A3 is located in the plasma membrane. Forskolin treatment increases the abundance of UT-A3 in the plasma membrane similar to UT-A1. However, these two transporters do not form a complex through a protein-protein interaction, suggesting that transporter function is unique to each protein. While immunohistochemistry localized UT-A3 to the basal and lateral membranes, a majority of the staining was cytosolic. Immunohistochemistry of vasopressin-treated rat kidney sections also localized UT-A3 primarily to the cytosol with basal and lateral membrane staining but also showed some apical membrane staining in some IMCD cells. This suggests that under normal conditions, UT-A3 functions as the basolateral transporter but in a high cAMP environment, the transporter may move from the cytosol to all plasma membranes to increase urea flux in the IMCD. In summary, this study confirms that UT-A3 is located in the inner medullary tip where it is expressed in the basolateral membrane, shows that UT-A3 is a phosphoprotein in rat IMCD that can be trafficked to the plasma membrane independent of UT-A1, and suggests that vasopressin may induce UT-A3 expression in the apical plasma membrane of IMCD. cAMP; vasopressin; concentrating mechanism; urea transport

The kidney responds to high levels of ANG II, as may occur during malignant hypertension, by increasing sodium and water excretion. To study whether kidney medullary transporters contribute to this response, rats were made hypertensive using ANG II. Within 3 days of being given ANG II, systolic blood pressure (BP) was increased (200 mmHg), vs control (130 mmHg), and remained high through day 14. Kidney inner medullary (IM) tip and base and outer medulla were analyzed for transporter protein abundance. There were significant decreases in UT-A1 urea transporter, aquaporin-2 (AQP2) water channel, and NKCC2/BSC1 [Na.sup.+]-[K.sup.+]-2[C1.sup.-] cotransporter. To determine whether the decreases were a response to hypertension, ANG II, or an ANG II-induced increase in aldosterone, rats were given 1) norepinephrine (to increase BP) and 2) ANG II plus spironolactone (to block the mineralocorticoid receptor). Norepinephrine (7 days) increased BP, urine volume, sodium excretion, and decreased urine osmolality and UT-A1, AQP2, and NKCC2/BSC1 abundances, similar to ANG II ANG II alone or with spironolactone yielded similar increases in BP, urine volume, and urine osmolality, and decreases in UT-A1 and AQP2 proteins in the IM tip. Plasma vasopressin was unaffected by treatment. Water diuresis did not change UT-A1 but decreased AQP2 and NKCC2/BSC1 abundances. We conclude that decreases in UT-A1, AQP2, and NKCC2/BSC1 proteins may contribute to the diuresis and natriuresis that occur following ANG II or norepinephrine-induced acute hypertension and do not appear to involve ANG II stimulation of aldosterone or thirst. aldosterone

Transepithelial [[sup.14]C]urea fluxes were measured across cultured Madin-Darby canine kidney (MDCK) cells permanently transfected to express the urea transport protein UT-A1. The urea fluxes were typically increased from a basal rate of 2 to 10 and 25 nmol x [cm.sup.-2] x [min.sup.-1] in the presence of vasopressin and forskolin, respectively. Flux activation consisted of a rapid-onset component of small amplitude that leveled off within ~ 10 min and at times even decreased again, followed by a delayed, strong increase over the next 30-40 min. Forskolin activated urea transport through activation of adenylyl cyclase; dideoxyforskolin was inactive. Vasopressin activated urea transport only from the basolateral side and was blocked by OPC-31260, indicating that its action was mediated by basolateral [V.sub.2] receptors. In the presence of the phosphodiesterase inhibitor IBMX, vasopressin activated as strongly as forskolin. By itself, IBMX caused a slow increase over 50 min to ~5 nmol x [cm.sup.-2] x [min.sup.-1] x 8-Bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP; 300 [micro]M) activated urea flux only when added basolaterally. IBMX augmented the activation by basolateral 8-BrcAMP. Urea flux activation by vasopressin and forskolin were only partially blocked by the protein kinase A inhibitor H-89. Even at concentrations >10 [micro]M, urea flux after 60 min of stimulation was reduced by urea transporter: Madin-Darby canine kidney cells

To identify novel gene targets of vasopressin regulation in the renal medulla, we performed a cDNA microarray study on the inner medullary tissue of mice following a 48-h water restriction protocol. In this study, 4,625 genes of the possible ~12,000 genes on the array were included in the analysis, and of these 157 transcripts were increased and 63 transcripts were decreased by 1.5-fold or more. Quantitative, real-time PCR measurements confirmed the increases seen for 12 selected transcripts, and the decreases were confirmed for 7 transcripts. In addition, we measured transcript abundance for many renal collecting duct proteins that were not represented on the array; aquaporin-2 (AQP2), AQP3, Pax-8, and [alpha]- and [beta]-Na-K-ATPase subunits were all significantly increased in abundance; the [beta]- and [gamma]-subunits of ENaC and the vasopressin type 1A receptor were significantly decreased. To correlate changes in mRNA expression with changes in protein expression, we carried out quantitative immunoblotting. For most of the genes examined, changes in mRNA abundances were not associated with concomitant protein abundance changes; however, AQP2 transcript abundance and protein abundance did correlate. Surprisingly, aldolase B transcript abundance was increased but protein abundance was decreased following 48 h of water restriction. Several transcripts identified by microarray were novel with respect to their expression in mouse renal medullary tissues. The steroid hormone enzyme 3[beta]-hydroxysteroid dehydrogenase 4 (3[beta]HSD4) was identified as a novel target of vasopressin regulation, and via dual labeling immunofluorescence we colocalized the expression of this protein to AQP2-expressing collecting ducts of the kidney. These studies have identified several transcripts whose abundances are regulated in mouse inner medulla in response to an increase in endogenous vasopressin levels and could play roles in the regulation of salt and water excretion. vasopressin; microarray

To investigate the underlying causes for aldose reductase deficiency-induced diabetes insipidus, we carried out studies with three genotypic groups of mice. These included wild-type mice, knockout mice, and a newly created bitransgenie line that was homozygous for both the aldose reductase null mutation and an aldose reductase knockin transgene driven by the kidney-specific cadherin promoter to direct transgene expression in the collecting tubule epithelial cells. We found that from early renal developmental stages onward, urine osmolality did not exceed 1,000 mosmol/kg[H.sub.2]O in aldose reductase-deficient mice. The functional defects were correlated with significant renal cellular and structural abnormalities that included cell shrinkage, apoptosis, disorganized tubular and vascular structures, and segmental atrophy. In contrast, the transgenic aldose reductase expression in the bitransgenic mice largely but incompletely rescued urine concentrating capacity and significantly improved renal cell survival, cellular morphology, and renal structures. Together, these results suggest that aldose reductase not only plays important roles in osmoregulation and medullary cell survival but may also be essential for the full maturation of the urine concentrating mechanism. urine concentration; diabetes insipidus

The primary mechanism by which the kidneys mediate net acid excretion is through ammonia metabolism. In the current study, we examined whether chronic metabolic acidosis, which increases ammonia metabolism, alters the cell-specific and/or the subcellular expression of the ammonia transporter family member, Rhcg, in the outer medullary collecting duct in the inner stripe (OMCDi). Chronic metabolic acidosis was induced in normal SD rats by HCl ingestion for 7 days; controls were pair-fed. The subcellular distribution of Rhcg was determined using immunogold electron microscopy and morphometric analyses. In intercalated cells, acidosis increased total Rhcg, apical plasma membrane Rhcg, and the proportion of total cellular Rhcg in the apical plasma membrane. Intracellular Rhcg decreased significantly, and basolateral Rhcg was unchanged. Because apical plasma membrane length increased in parallel with apical Rhcg immunolabel, apical plasma membrane Rhcg density was unchanged. In principal cells, acidosis increased total Rhcg, apical plasma membrane Rhcg, and the proportion of total cellular Rhcg in the apical plasma membrane while decreasing the intracellular proportion. In contrast to the intercalated cell, chronic metabolic acidosis did not significantly alter apical boundary length; accordingly, apical plasma membrane Rhcg density increased. In addition, basolateral Rhcg immunolabel increased in response to chronic metabolic acidosis. These results indicate that in the rat OMCDi 1) chronic metabolic acidosis increases apical plasma membrane Rhcg in both the intercalated cell and principal cell where it may contribute to enhanced apical ammonia secretion; 2) increased apical plasma membrane Rhcg results from both increased total protein and changes in the subcellular distribution of Rhcg; 3) the mechanism of Rhcg subcellular redistribution differs in intercalated and principal cells; and 4) Rhcg may contribute to regulated basolateral ammonia transport in the principal cell. intercalated cell; principal cell; immunogold; morphometry

Mammalian urea transporters are facilitated membrane transport proteins belonging to two families, UT-A and UT-B. They are best known for their role of maintaining the renal inner medullary urinary concentrating gradient. Urea transporters have also been identified in tissues not typically associated with urea metabolism. The purpose of this study was to survey the major organs in rat to determine the distribution of UT-A and UT-B mRNA transcripts and protein forms and determine their cellular localization. Five kidney subregions and 17 extrarenal tissues were screened by Northern blot analysis using two UT-A and three UT-B probes and by Western blot analysis using polyclonal COOH-terminal UT-A and UT-B antibodies. Immunohistochemistry was performed on 16 extrarenal tissues using the same antibodies. In kidney, we detected mRNA transcripts and protein bands consistent with previously-identified UT-A and UT-B isoforms, as well as novel forms. We found that UT-A mRNA and protein are widely expressed in extrarenal tissues in various forms that are different from the known isoforms. We determined the cellular localization of UT-A and UT-B in these tissues. We found that both UT-A and UT-B are ubiquitously expressed as numerous tissue-specific mRNA transcripts and protein forms that are localized to cell membranes, cytoplasm, or nuclei. urea transporter; Northern blot analysis; Western blot analysis; immunohistochemistry

Chronic metabolic acidosis indramatic increases in net acid excretion that are predomidue to increases in urinary ammonia excretion. The current study examines whether this increase is associated with changes in the expression of the renal ammonia transporter family members, Rh B glycoprotein (Rhbg) and Rh C glycoprotein (Rhcg). Chronic metabolic acidosis was induced in Sprague-Dawley rats by HC1 ingestion for 1 wk; control animals were pair-fed. After 1 wk, metabolic acidosis had developed, and urinary ammonia excretion increased significantly. Rhcg protein expression was increased in both the outer medulla and the base of the inner medulla. Intercalated cells in the outer medullary collecting duct (OMCD) and in the inner medullary collecting duct (IMCD) in acid-loaded animals protruded into the tubule lumen and had a sharp, discrete band of apical Rhcg immunoreactivity, compared with a flatter cell profile and a broad band of apical immunolabel in control kidneys. In addition, basolateral Rhcg immunoreactivity was observed in both control and acidotic kidneys. Cortical Rhcg protein expression and immunoreactivity were not detectably altered. Rhcg mRNA expression was not significantly altered in the cortex, outer medulla, or inner medulla by chronic metabolic acidosis. Rhbg protein and mRNA expression were unchanged in the cortex, outer and inner medulla, and no changes in Rhbg immunolabel were evident in these regions. We conclude that chronic metabolic acidosis increases Rhcg protein expression in intercalated cells in the OMCD and in the IMCD, where it is likely to mediate an important role in the increased urinary ammonia excretion. connecting segment; collecting duct

In normal rats, vasopressin and hyperosmolality enhance urea permeability ([P.sub.urea]) in the terminal, but not in the initial inner medullary collecting duct (IMCD), a process thought to occur through the UT-A1 urea transporter. In the terminal IMCD, UT-A1 is detected as 97- and 117-kDa glycoproteins. However, in the initial IMCD, only the 97-kDa form is detected. During streptozotocin-induced diabetes mellitus, UT-A1 protein abundance is increased, and the 117-kDa UT-A 1 glycoprotein appears in the initial IMCD. We hypothesize that the 117-kDa glycoprotein mediates the vasopressin- and osmolality-induced changes in [P.sub.urea]. Thus, in the present study, we measured [P.sub.urea] in in vitro perfused initial IMCDs from diabetic rats by imposing a 5 mM bath-to-lumen urea gradient without any osmotic gradient. Basal [P.sub.urea] was similar in control vs. diabetic rats (3 [+ or -] 1 vs. 5 [+ or -] 1 x [10.sup.-5] cm/s, n = 4, P = not significant). Vasopressin (10 [micro]M) significantly increased [P.sub.urea] to 16 [+ or -] 5 x [10.sup.-5] cm/s (n = 4, P < 0.05) in diabetic but not in control rats. Forskolin (10 [micro]M, adenylyl cyclase activator) also significantly increased [P.sub.urea] in diabetic rats. In contrast, increasing osmolality to 690 mosmol/kg[H.sub.2]O did not change [P.sub.urea] in diabetic rats. We conclude that initial IMCDs from diabetic rats have vasopressin- and forskolin-, but not hyperosmolality-stimulated [P.sub.urea]. The appearance of vasopressin-stimulated [P.sub.urea] in initial IMCDs correlates with an increase in UT-A1 protein abundance and the appearance of the 117-kDa UT-A1 glycoprotein in this region during diabetes. This suggests that the 117-kDa UT-A1 glycoprotein is necessary for vasopressin-stimulated urea transport. diabetes mellitus; inner medullary collecting duct; hyperosmolality

Production, recycling, and elimination of urea are important to maintain nitrogen balance. Adaptation to varying loads of urea due to different protein intake or in renal failure may involve changes in urea transport and may possibly affect urea transporters. In this study, we examined the expression of the UT-B urea transporter in rats fed a low-protein diet (LPD), a high-protein diet (HPD), and a 20% urea-supplemented diet. In the kidney, UT-B protein abundance increased in the outer medulla of both LPD-fed rats and 20% urea-fed rats, without changes in the inner medulla of either group compared with controls. In HPD-fed rats, UT-B protein decreased significantly in both the outer and inner medulla. We identified expression of UT-B in the rat colon, as a 2-kb mRNA transcript and as an ~45-kDa protein, with apical localization in superficial colon epithelial cells. UT-B also is expressed in rat small intestine. In rat colon, UT-B protein abundance was mildly, but significantly, decreased in LPD-fed and 20% urea-fed rats. UT-B abundance also was examined in the colon of 7/8 nephrectomized, uremic rats and in HPD-fed rats and was not significantly different from that in control rats. These findings indicate that UT-B expression is regulated in response to different loads of urea, with a pattern that suggests involvement of tissue-specific regulatory mechanism in kidney and colon. kidney; urea transport; colon; uremia

Rats with diabetes mellitus have an increase in UT-A1 urea transporter protein abundance and absolute urea excretion, but the relative amount (percentage) of urea in total urinary solute is actually decreased due to the marked glucosuria. Urea-specific signaling pathways have been identified in mIMCD3 cells and renal medulla, suggesting the possibility that changes in the percentage or concentration of urea could be a factor that regulates UT-A1 abundance. In this study, we tested the hypothesis that an increase in a urinary solute other than urea would increase UT-A1 abundance, similar to diabetes mellitus, whereas an increase in urine urea would not. In both inner medullary base and tip, UT-A1 protein abundance increased during NaCl- or glucose-induced osmotic diuresis but not during urea-induced osmotic diuresis. Next, rats undergoing NaCl or glucose diuresis were given supplemental urea to increase the percentage of urine urea to control values. UT-A1 abundance did not increase in these urea-supplemented rats compared with control rats. Additionally, both UT-A2 and UT-B protein abundances in the outer medulla increased during urea-induced osmotic diuresis but not in NaCl or glucose diuresis. We conclude that during osmotic diuresis, UT-A1 abundance increases when the percentage of urea in total urinary solute is low and UT-A2 and UT-B abundances increase when the urea concentration in the medullary interstitium is high. These findings suggest that a reduction in urine or interstitial urea results in an increase in UT-A1 protein abundance in an attempt to restore inner medullary interstitial urea and preserve urine-concentrating ability. sodium-potassium-2 chloride cotransporter; diabetes mellitus

We have identified a urea transporter from the mucosa of the human colon that has characteristics consistent with a Kidd antigen/UT-B urea transporter. This intestinal urea transporter encodes a 389-amino acid peptide with a sequence identical to that previously reported for the UT-B urea transporter in erythrocytes. Expression of a UT-B 2-kb mRNA transcript and of ~50- and ~98-kDa UT-B proteins is detected in human colonic mucosa by Northern and Western blot analysis. The UT-B protein is localized in the cell membrane and cytoplasm of the superficial intestinal epithelium and in the epithelial cells in the crypts. A 2-kb UT-B mRNA transcript and the UT-B protein were also identified in the intestinal cell line Caco-2. The transepithelial flux of [sup.14]C urea was examined in Caco-2 cells growing on porous membrane support and was significantly inhibited by phloretin, 1,3-dimethylurea, and thiourea, suggesting that the transfer of urea across the Caco-2 monolayer could be mediated, at least in part, by the UT-B urea transporter. We conclude that the Kidd antigen/UT-B urea transporter is physiologically expressed in the human colon epithelium, where it could participate in the transport of urea across the colon mucosa. urea transport; Caco-2

Altered expression of urea transporters in response to ureteral obstruction. Am J Physiol Renal Physiol 286:F1154-F1162, 2004. First published February 24, 2004; 10.1152/ajprenal.00453.2003.--Urea plays an important role in the urinary concentrating capacity. Renal inner medullary (IM) urea transporter expression was examined in rats with bilateral (BUO) or unilateral ureteral obstruction (UUO). BUO (24 h) was associated with markedly increased plasma urea (42.4 [+ or -] 1.0 vs. 5.2 [+ or -] 0.2 mmol/l) and a significant decease in expression of UT-A1 (28 [+ or -] 8% of sham levels), UT-A3 (45 [+ or -] 11%), and UT-B (70 [+ or -] 8%). Immunocytochemistry confirmed downregulation of UT-Al and UT-A3 in IM collecting duct and UT-B in the descending vasa recta. Three days after release of BUO, UT-A1, UT-A3, and UT-B remained significantly dowuregulated (UT-A1: 37 [+ or -] 6%; UT-A3:25 [+ or -] 6%; and UT-B: 10 [+ or -] 5% of sham levels; P < 0.05) concurrent with a persistent polyuria and a marked reduction in solute-free water reabsorption (115 [+ or -] 11 vs. 196 [+ or -] 8 [micro]l x [min.sup.-1] x [kg.sup.-1] p < 0.05). Moreover, 14 days after release of BUO, total UT-Al, UT-A3, and UT-B remained significantly decreased compared with sham-operated controls and urine urea remained reduced (588 [+ or -] 43 vs. 1,150 [+ or -] 94 mmol/l). Consistent with increased levels of plasma urea 24 h after onset of UUO (7.4 [+ or -] 0.3 vs. 4.8 [+ or -] 0.3 mmol/l), the protein abundance of UT-A1, UT-A3, and UT-B in IM was markedly reduced in the obstructed kidney, which was confirmed by immunocytochemistry. In the nonobstructed kidney, the expression of urea transporters did not change. In conclusion, reduced expression of UT-A1, UT-A3, and UT-B levels in both BUO and UUO rats suggests that urea transporters play important roles in the impaired urinary concentrating capacity in response to urinary tract obstruction. collecting duct; descending vasa recta; obstructive nephropathy; urine concentrating mechanism

Urea transport in MDCK cells that are stably transfected with UT-A1. Am J Physiol Cell Physiol 286: C1264-C1270, 2004. First published February 4, 2004; 10.1152/ ajpcelh00499.2003.--Progress in understanding the cell biology of urea transporter proteins has been hampered by the lack of an appropriate cell culture system. The goal of this study was to create a polarized epithelial cell line that stably expresses the largest of the rat renal urea transporter UT-A isoforms, UT-A1. The gene for UT-A1 was cloned into pcDNA5/FRT and transfected into Madin-Darby canine kidney (MDCK) cells with an integrated Flp recombination target site. The cells from a single clone were grown to confluence on collagen-coated membranes until the resistance was > 1,500 [ohm].[cm.sup.2]. Transepithelial [[.sub.14]C]urea fluxes were measured at 37[degrees]C in a HC[O.sup.-.sub.3]/ C[O.sub.2] buffer, pH 7.4, with 5 mM urea. The baseline fluxes were not different between unstimulated UT-Al-transfected MDCK cells and nontransfected or sham-transfected MDCK cells. However, only in the UT-Al-transfected cells was UT-Al protein expressed (as measured by Western blot analysis) and urea transport stimulated by forskolin or arginine vasopressin. Forskolin and arginine vasopressin also increased the phosphorylation of UT-Al. Thionicotinamide, dimethylurea, and phloretin inhibited the forskolin-stimulated [[.sup.14]C]-urea fluxes in the UT-A1-transfected MDCK cells. These characteristics mimic those seen in rat terminal inner medullary collecting ducts. This new polarized epithelial ceil line stably expresses UT-Al and reproduces several of the physiological responses observed in rat terminal inner medullary collecting ducts. urea transporter-A1; arginine vasopressin; collecting duct; Madin-Darby canine kidney cells

Kim, Dongun, Jeff M. Sands, and Janet D. Klein. Role of vasopressin in diabetes mellitus-induced changes in medullary transport proteins involved in urine concentration in Brattleboro rats. Am J Physiol Renal Physiol 286: F760-F766, 2004. First published November 25, 2003; 10.1152/ajprenal.00369.2003.--In rats with streptozotocin-induced diabetes mellitus for 10-20 days, we showed that the abundance of the major medullary transport proteins involved in the urinary concentrating mechanism, urea transporter (UT-A1), aquaporin-2 (AQP2), and the [Na.sup.+]-[K.sup.+]-2[C1.sup.-] cotransporter (NKCC2/ BSC1), is increased, despite the ongoing osmotic diuresis. To test whether vasopressin is necessary for these diabetes mellitus-induced changes in UT-A1, AQP2, or NKCC2/BSC1, we studied Brattleboro rats because they lack vasopressin. Brattleboro rats were given vasopressin (2.4 [micro]g/day via osmotic minipump) for 5 or 12 days. At 5 days, vasopressin increased AQP2 protein abundance but decreased UT-A1 abundance compared with untreated Brattleboro rats. At 12 days, vasopressin increased the abundance of both UT-A1 and AQP2 proteins but did not alter NKCC2/BSC1. Next, untreated Brattleboro rats were made diabetic for 10 days by injecting them with streptozotocin (40 mg/kg). Diabetes mellitus increased the abundance of AQP2 and NKCC2/BSC1 proteins, but UT-A1 protein abundance did not increase. Third, vasopressin-treated Brattleboro rats were made diabetic with streptozotocin for 10 days. In vasopressin-treated Brattleboro rats, diabetes mellitus increased UT-A1, AQP2, and NKCC2/BSC1 protein abundances. Vasopressin significantly increased UT-A1 phosphorylation in vasopressin-treated diabetic Brattleboro rats but not in the other groups of Brattleboro rats. We conclude that 1) administering vasopressin to Brattleboro rats for 12 days, but not for 5 days, increases UT-A1 protein abundance and 2) vasopressin is necessary for the increase in UT-A1 protein in diabetic rats but is not necessary for the increase in AQP2 or NKCC2 proteins. urea transporter; aquaporin; urinary concentrating ability; sodium/ potassium/chloride cotransport

We tested whether the abundance of transport proteins involved in the urinary concentrating mechanism was altered in rats with uncontrolled diabetes mellitus (DM). Rats were injected with streptozotocin and killed 5, 10, 14, or 20 days later. Blood glucose in DM rats was 300-450 mg/dl (control: 70-130 mg/dl). Urine volume increased in DM rats from 41 [+ or -] 7 ml/100 g body wt (BW) at 5 days to 69 [+ or -] 3 ml/100 g BW at 20 days (control: 9 [+ or -] 1). Urine osmolality of DM rats decreased at 5 days DM and remained low at 20 days. UT-A1 urea transporter protein in the inner medullary (IM) tip was 55% of control in 5-day DM rats but increased to 170,220, and 280% at 10, 14, and 20 days DM, respectively, due to an increase in the 117-kDa glycoprotein form. UT-A1 in the IM base was increased to 325% of control at 5 days DM with no further increase at 20 days. Aquaporin-2 (AQP2) increased to 290% in the IM base at 5 days DM and 150% in the IM tip at 10 days; both showed no further increase at 20 days. NKCC2/ BSC1 increased to 240% in outer medulla at 20 days DM, but not at 5 or 10 days. UT-B and ROMK were unchanged at any time point. The increases in UT-A1, AQP2, and NKCC2/ BSC1 proteins during uncontrolled DM would tend to limit the loss of fluid and solute during uncontrolled diabetes. urea; water; urea transporter; aquaporin; sodium

Our laboratory previously reported that uremic levels of urea inhibit L-arginine (L-Arg) transport into endothelial cells. The present study further investigated this effect. We measured L-Arg transport in cultured bovine aortic endothelial cells with normal or high urea (25 mM). The urea transport inhibitor phloretin abolished the inhibitory effect of urea on L-Arg transport, suggesting a role for urea transporters (UTs). We screened bovine aortic endothelial cells and several other endothelial cell types for the presence of UTs by using Western blot analysis. UT-B was present in all endothelial cells, irrespective of species or location of derivation, whereas UT-A distribution was variable and sparse. UT-B was also abundant in rat aorta, mesenteric blood vessels, and spinotrapezius muscle, whereas UT-A distribution was, again, variable and sparse. Chronic elevation of urea had variable, inconsistent effects on UT abundance. This study showed that urea must enter endothelial cells, probably by UT-B, to inhibit L-Arg transport. In view of the wide distribution of UT-B in rat vasculature, elevated blood urea nitrogen may lead to endothelial L-Arg deficiency in vivo. uremia; nitric oxide; phloretin; blood vessels

ACE.2 mice lack all tissue angiotensin-converting enzyme (ACE) but have 33% of normal plasma ACE activity. They exhibit the urine-concentrating defect and hyperkalemia present in mice that lack all ACE, but in contrast to the complete knockout, ACE.2 mice have normal medullary histology and creatinine clearance. To explore the urine-concentrating defect in ACE.2 mice, renal medullary transport proteins were analyzed using Western blot analysis. In the inner medulla, UT-A1, ClC-K1, and aquaporin-1 (AQP1) were significantly reduced to 28 [+ or-] 5, 6 [+ or-] 6, and 39 [+ or-] 5% of the level in wild-type mice, respectively, whereas AQP2 and UT-B were unchanged. In the outer medulla, [Na.sup.+]-[K.sup.+]-2[Cl.sup.-] cotransporter (NKCC2/BSC1) and AQP1 were significantly reduced to 56 [+ or-] 11 and 29 [+ or-] 6%, respectively, whereas [Na.sup.+]-[K.sup.+]-ATPase, UT-A2, UT-B, and AQP2 were unchanged, and renal outer medullary potassium channel was significantly increased to 711 [+ or-] 187% of the level in wild-type mice. The abnormal expression of these transporters was similar in ACE.2 mice backcrossed onto a C57BL/6 or a Swiss background and was not rescued by ANG II infusion. We conclude that the urine-concentrating defect in ACE.2 mice is associated with, and may result from, downregulation of some or all of these key urea, salt, and water transport proteins. urine-concentrating mechanism; angiotensin; urea; sodium chloride; potassium; aquaporin; angiotensin-converting enzyme

Vasopressin rapidly increases phosphorylation of UT-A1 urea transporter in rat IMCDs through PICA. Am J Physiol Renal Physiol 282: F85-F90, 2002. First published August 8, 2001; 10.1152/ajprenal.00054.2001.--The UT-A1 urea transporter plays an important role in maintaining the hyperosmolar milieu of the inner medulla. Vasopressin increases urea permeability in rat terminal inner medullary collecting ducts (IMCDs) within 5-10 min. To elucidate the mechanism, IMCD suspensions were radiolabeled with [[sup.32]P]orthophosphate. UT-A1 was immunoprecipitated and analyzed by autoradiogram and Western blot. Both the 97- and 117-kDa UT-A1 proteins were phosphorylated. Vasopressin treatment increased the phosphorylation of both UT-A1 proteins at 2 min, which peaked at 5-10 min and remained elevated for up to 30 min. There was a discernable increase in UT-A1 phosphorylation with 10 pM and a 50% increase with 10-100 nM vasopressin, 1-Desamino-8-D-arginine vasopressin (dDAVP) or 8-(4-chlorophenylthio)-cAMP (CPT-cAMP) also increased UT-A1 phosphorylation. The vasopressin-stimulated increase in UT-A1 phosphorylation was blocked by H-89 or a specific peptide inhibitor of protein kinase A. Phosphatase inhibitors (okadaic acid, calyculin) increased UT-A1 phosphorylation. We conclude that vasopressin increases UT-A1 phosphorylation via protein kinase A within 2-5 min in rat IMCDs. This suggests that phosphorylation of UT-A1 may be the mechanism by which vasopressin rapidly increases urea permeability in vivo. [V.sub.2] receptor; adenosine 3',5'-cyclic monophosphate; urine-concentrating mechanism; urea permeability; inner medullary collecting duct; protein kinase A

Jiang, Gengru, Janet D. Klein, and W. Charles O'Neill. Growth factors stimulate the Na-K-2C1 cotransporter NKCC1 through a novel Cl--dependent mechanism. Am J Physiol Cell Physiol 281: C1948-C1953, 2001.--The Na-K-2C1 cotransporter NKCC1 is an important volume-regulatory transporter that is regulated by cell volume and intracellular [Cl.sup.-]. This regulation appears to be mediated by phosphorylation of NKCC1, although there is evidence for additional, cytoskeletal regulation via myosin light chain (MLC) kinase. NKCC1 is also activated by growth factors and may contribute to cell hypertrophy, but the mechanism is unknown. In aortic endothelial cells, NKCC1 (measured as bumetanide-sensitive [sup.86.Rb.sup.+] influx) was rapidly stimulated by serum, lysophosphatidic acid, and fibroblast growth factor, with the greatest stimulation by serum. Serum increased bumetanide-sensitive influx significantly more than bumetanide-sensitive efflux (131% vs. 44%), indicating asymmetric stimulation of NKCC1, and produced a 17% increase in cell volume and a 25% increase in [Cl.sup.-] content over 15 min. Stimulation by serum and hypertonic shrinkage were additive, and serum did not increase phosphorylation of NKCC1 or MLC, and did not decrease cellular [Cl.sup.-] content. When cellular [Cl.sup.-] was replaced with methanesulfonate, influx via NKCC1 increased and was no longer stimulated by serum, whereas stimulation by hypertonic shrinkage still occurred. Based on these results, we propose a novel mechanism whereby serum activates NKCC1 by reducing its sensitivity to inhibition by intracellular [Cl.sup.-]. This resetting of the [Cl.sup.-] set point of the transporter enables the cotransporter to produce a hypertrophic volume increase. Na-K-2CI cotransport; serum; lysophosphatidic acid; fibroblast growth factor; intracellular chloride; vascular endothelium Received 11 Januart 2001; accepted in final form 27 July 2001

The activity of Na+-K+-2Cl- cotransporter NKCC1 in vascular smooth muscle was measured as the bumetanide-sensitive efflux of 86Rb+ from intact smooth muscle of the rat aorta. Results showed that the Na+-K+-2Cl- cotransporter in vascular smooth muscle is reciprocally controlled by vasoconstrictors and nitrovasodilators and contributes to smooth muscle contraction, indicating that changes in NKCC1 could affect vascular smooth muscle tone in vivo.

Research shows that biochemical vasopressin-regulated urea transporter (VRUT)protein is controlled by biochemical glucocorticoids in a pathway separate from that involving insulin. Experiments using diabetic male Sprague-Dawley rats are described. VRUT protein levels are lower in adrenalectomized rats and rats with diabetes mellitus.

The mechanisms the mediate the shrinkage induced activation of the sodium-hydrogen exchanger (NHE) in C6 cells were analyzed to determine its similarity of the shrinkage-induced activation of NHE in astrocytes. The exposure of C6 glioma cells to hyperosmotic solutions induced the activation of NHE in a manner that was directly proportional to shrinkage. Furthermore, shrinkage-induced activation of NHE in the C6 glioma cells was mediated by myosin light chain kinase.

A study using hypertonically shrunken aortic endothelial cells, immunoblotting and immune precipitation was conducted to determine the mechanism by which cells activate the sodium ion/potassium ion/chloride (Na-K-Cl) cotransporter in response to changes in cell volume. The results show that a protein kinase phosphorylates a 19-kiloDalton cytoskeletal protein called myosin light chain. The protein kinase affects Na-K-Cl cotransporter activity independently of cotransporter phosphorylation.

Volume homeostasis is very important especially in brain cells and astrocytes are the most important brain cell type. Volume changes in this type of cell can alter the relationship with other parts of the brain and lead to neurological impairment. Shrinkage-induced activation of Na+/ H+ exchange (NHE) in astrocytes is mediated by a novel activation pathway involving calmodulin-dependent myosin light chain kinase and phosphorylation of myosin light chain. Ca2+, cAMP, or PKC is uninvolved in the shrinkage induced activation of NHE.

14-3-3γ is a small protein regulating its target proteins through binding to phosphorylated serine/threonine residues. Sequence analysis of large-conductance Ca2+-activated K+ (BK) channels revealed a putative 14-3-3 binding site in the COOH-terminal region. Our previous data showed that 14-3-3γ is widely expressed in the mouse kidney. Therefore, we hypothesized that 14-3-3γ has a novel role in the regulation of BK channel activity and protein expression. We used electrophysiology, Western blot analysis, and coimmunoprecipitation to examine the effects of 14-3-3γ on BK channels both in vitro and in vivo. We demonstrated the interaction of 14-3-3γ with BK α-subunits (BKα) by coimmunoprecipitation. In human embryonic kidney-293 cells stably expressing BKα, overexpression of 14-3-3γ significantly decreased BK channel activity and channel open probability. 14-3-3γ inhibited both total and cell surface BKα protein expression while enhancing ERK1/2 phosphorylation in Cos-7 cells cotransfected with flag-14-3-3γ and mycBK. Knockdown of 14-3-3γ by siRNA transfection markedly increased BKα expression. Blockade of the ERK1/2 pathway by incubation with the MEK-specific inhibitor U0126 partially abolished 14-3-3γ-mediated inhibition of BK protein expression. Similarly, pretreatment of the lysosomal inhibitor bafilomycin A1 reversed the inhibitory effects of 14-3-3γ on BK protein expression. Furthermore, overexpression of 14-3-3γ significantly increased BK protein ubiquitination in embryonic kidney-293 cells stably expressing BKα. Additionally, 3 days of dietary K+ challenge reduced 14-3-3γ expression and ERK1/2 phosphorylation while enhancing renal BK protein expression and K+ excretion. These data suggest that 14-3-3γ modulates BK channel activity and protein expression through an ERK1/2-mediated ubiquitin-lysosomal pathway. [ABSTRACT FROM AUTHOR]

Renal fibrosis is a major contributor to the development and progression of chronic kidney disease. A low-protein diet can reduce the progression of chronic kidney disease and reduce the development of renal fibrosis, although the mechanism is not well understood. Urea reabsorption into the inner medulla is regulated by inner medullary urea transporter (UT)-A1 and UT-A3. Inhibition or knockout of UT-A1/A3 will reduce interstitial urea accumulation, which may be beneficial in reducing renal fibrosis. To test this hypothesis, the effect of unilateral ureteral obstruction (UUO) was compared in wild-type (WT) and UT-A1/A3 knockout mice. UUO causes increased extracellular matrix associated with increases in transforming growth factor-β, vimentin, and α-smooth muscle actin (α-SMA). In WT mice, UUO increased the abundance of three markers of fibrosis: transforming growth factor-β, vimentin, and α-SMA. In contrast, in UT-A1/A3 knockout mice, the increase following UUO was significantly reduced. Consistent with the Western blot results, immunohistochemical staining showed that the levels of vimentin and α-SMA were increased in WT mice with UUO and that the increase was reduced in UT-A1/A3 knockout mice with UUO. Masson’s trichrome staining showed increased collagen in WT mice with UUO, which was reduced in UT-A1/A3 knockout mice with UUO. We conclude that reduced UT activity reduces the severity of renal fibrosis following UUO. [ABSTRACT FROM AUTHOR]

The transient receptor potential canonical 6 (TRPC6) channel and podocin are colocalized in the glomerular slit diaphragm as an important complex to maintain podocyte function. Gain of TRPC6 function and loss of podocin function induce podocyte injury. We have previously shown that high glucose induces apoptosis of podocytes by activating TRPC6; however, whether the activated TRPC6 can alter podocin expression remains unknown. Western blot analysis and confocal microscopy were used to examine both expression levels of TRPC6, podocin, and nephrin and morphological changes of podocytes in response to high glucose. High glucose increased the expression of TRPC6 but reduced the expression of podocin and nephrin, in both cultured human podocytes and type 1 diabetic rat kidneys. The decreased podocin was diminished in TRPC6 knockdown podocytes. High glucose elevated intracellular Ca2+ in control podocytes but not in TRPC6 knockdown podocytes. High glucose also elevated the expression of a tight junction protein, zonula occludens-1, and induced the redistribution of zonula occludens-1 and loss of podocyte processes. These data together suggest that high glucose reduces protein levels of podocin by activating TRPC6 and induces morphological changes of cultured podocytes. [ABSTRACT FROM AUTHOR]