Janet D. Klein

Changes in aquaporin-2 protein contribute to the urine concentrating defect in rats fed a low-protein diet.

Low-protein diets cause a urinary concentrating defect in rats and humans. Previously, we showed that feeding rats a low (8%) protein diet induces a change in urea transport in initial inner medullary collecting ducts (IMCDs) which could contribute to the concentrating defect. Now, we test whether decreased osmotic water permeability (Pf) contributes to the concentrating defect by measuring Pf in perfused initial and terminal IMCDs from rats fed 18 or 8% protein for 2 wk. In terminal IMCDs, arginine vasopressin (AVP)-stimulated osmotic water permeability was significantly reduced in rats fed 8% protein compared to rats fed 18% protein. In initial IMCDs, AVP-stimulated osmotic water permeability was unaffected by dietary protein. Thus, AVP-stimulated osmotic water permeability is significantly reduced in terminal IMCDs but not in initial IMCDs. Next, we determined if the amount of immunoreactive aquaporin-2 (AQP2, the AVP-regulated water channel) or AQP3 protein was altered. Protein was isolated from base or tip regions of rat inner medulla and Western analysis performed using polyclonal antibodies to rat AQP2 or AQP3 (courtesy of Dr. M.A. Knepper, National Institutes of Health, Bethesda, MD). In rats fed 8% protein (compared to rats fed 18% protein): (a) AQP2 decreases significantly in both membrane and vesicle fractions from the tip; (b) AQP2 is unchanged in the base; and (c) AQP3 is unchanged. Together, the results suggest that the decrease in AVP-stimulated osmotic water permeability results, at least in part, in the decrease in AQP2 protein. We conclude that water reabsorption, like urea reabsorption, responds to dietary protein restriction in a manner that would limit urine concentrating capacity.

Phosphorylation of UT-A1 urea transporter at serines 486 and 499 is important for vasopressin-regulated activity and membrane accumulation

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-PK1 cells and increased urea flux in oocytes. In contrast, the S486A and S499A mutants demonstrated loss of forskolin-stimulated UT-A1 phosphorylation and reduced urea flux. In LLC-PK1 cells, we assessed biotinylated UT-A1. Wild-type UT-A1, S486A, and S499A accumulated in the membrane in response to forskolin. However, in the S486A/S499A double mutant, forskolin-stimulated 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.

Vasopressin Increases Plasma Membrane Accumulation of Urea Transporter UT-A1 in Rat Inner Medullary Collecting Ducts

Urea transport, mediated by the urea transporter A1 (UT-A1) and/or UT-A3, is important for the production of concentrated urine. Vasopressin rapidly increases urea transport in rat terminal inner medullary collecting ducts (IMCD). A previous study showed that one mechanism for rapid regulation of urea transport is a vasopressin-induced increase in UT-A1 phosphorylation. This study tests whether vasopressin or directly activating adenylyl cyclase with forskolin also increases UT-A1 accumulation in the plasma membrane of rat IMCD. Inner medullas were harvested from rats 45 min after injection with vasopressin or vehicle. UT-A1 abundance in the plasma membrane was significantly increased in the membrane fraction after differential centrifugation and in the biotinylated protein population. Vasopressin and forskolin each increased the amount of biotinylated UT-A1 in rat IMCD suspensions that were treated ex vivo. The observed changes in the plasma membrane are specific, as the amount of biotinylated UT-A1 but not the calcium-sensing receptor was increased by forskolin. Next, whether forskolin or the V(2)-selective agonist dDAVP would increase apical membrane expression of UT-A1 in MDCK cells that were stably transfected with UT-A1 (UT-A1-MDCK cells) was tested. Forskolin and dDAVP significantly increased UT-A1 abundance in the apical membrane in UT-A1-MDCK cells. It is concluded that vasopressin and forskolin increase UT-A1 accumulation in the plasma membrane in rat IMCD and in the apical plasma membrane of UT-A1-MDCK cells. These findings suggest that vasopressin regulates urea transport by increasing UT-A1 accumulation in the plasma membrane and/or UT-A1 phosphorylation.

Exercise ameliorates chronic kidney disease-induced defects in muscle protein metabolism and progenitor cell function.

Chronic kidney disease (CKD) impairs muscle protein metabolism leading to muscle atrophy, and exercise can counteract this muscle wasting. Here we evaluated how resistance exercise (muscle overload) and endurance training (treadmill running) affect CKD-induced abnormalities in muscle protein metabolism and progenitor cell function using mouse plantaris muscle. Both exercise models blunted the increase in disease-induced muscle proteolysis and improved phosphorylation of Akt and the forkhead transcription factor FoxO1. Muscle overloading, but not treadmill running, corrected protein synthesis and levels of mediators of protein synthesis such as phosphorylated mTOR and p70S6K in the muscles of mice with CKD. In these mice, muscle overload, but not treadmill, running, increased muscle progenitor cell number and activity as measured by the amounts of MyoD, myogenin, and eMyHC mRNAs. Muscle overload not only increased plantaris weight and reduced muscle proteolysis but also corrected intracellular signals regulating protein and progenitor cell function in mice with CKD. Treadmill running corrects muscle proteolysis but not protein synthesis or progenitor cell function. Our results provide a basis for evaluating different types of exercise on muscle atrophy in patients with chronic kidney disease.

JNK is a volume-sensitive kinase that phosphorylates the Na-K-2Cl cotransporter in vitro

Cell shrinkage phosphorylates and activates the Na-K-2Cl cotransporter (NKCC1), indicating the presence of a volume-sensitive protein kinase. To identify this kinase, extracts of normal and shrunken aortic endothelial cells were screened for phosphorylation of NKCC1 fusion proteins in an in-the-gel kinase assay. Hypertonic shrinkage activated a 46-kDa kinase that phosphorylated an NH2-terminal fusion protein, with weaker phosphorylation of a COOH-terminal fusion protein. This cytosolic kinase was activated by both hypertonic and isosmotic shrinkage, indicating regulation by cell volume rather than osmolarity. Subsequent studies identified this kinase as c-Jun NH2-terminal kinase (JNK). Immunoblotting revealed increased JNK activity in shrunken cells; there was volume-sensitive phosphorylation of NH2-terminal c-Jun fusion protein; immunoprecipitation of JNK from shrunken cells but not normal cells phosphorylated NKCC1 in gel kinase assays; and treatment of cells with tumor necrosis factor, a known activator of JNK, mimicked the effect of hypertonicity. We conclude that JNK is a volume-sensitive kinase in endothelial cells that phosphorylates NKCC1 in vitro. This is the first demonstration of a volume-sensitive protein kinase capable of phosphorylating a volume-regulatory transporter.

Upregulation of Urea Transporter UT-A2 and Water Channels AQP2 and AQP3 in Mice Lacking Urea Transporter UT-B

The UT-B urea transporter is the major urea transporter in red blood cells and kidney descending vasa recta. Humans and mice that lack UT-B have a mild urine-concentrating defect. Whether deletion of UT-B altered the expression of other transporter proteins involved in urinary concentration was tested. Fluorescence-based real-time reverse transcription-PCR and Northern blot analysis showed upregulation of the UT-A2 urea transporter and the aquaporin 2 (AQP2) and AQP3 water channel transcripts but no change in other urea transporters or AQP. Western blot analysis showed that UT-A2 protein abundance in the outer medulla of UT-B null mice increased to 122 +/- 6% of wild-type control. AQP2 protein abundance increased to 177 +/- 32% and 127 +/- 7% in the outer and inner medulla, respectively, of UT-B null versus wild-type mice. The abundance of UT-A1, AQP1, renal outer medullary potassium channel, and NKCC2/BSC1 proteins were not significantly different between UT-B null and wild-type mice. The increases in AQP2 and AQP3 would reduce water loss and improve concentrating ability. The lack of UT-B does not result in a change in expression of urea transporters involved in urea reabsorption from the inner medullary collecting duct (UT-A1 and UT-A3). However, UT-B null mice have a selective increase in UT-A2 protein abundance. This may be an adaptive response to the loss of UT-B, because UT-B and UT-A2 are involved in different intrarenal urea recycling pathways.

Regulation by cell volume of Na+-K+-2Cl− cotransport in vascular endothelial cells: role of protein phosphorylation

SummaryNa+-K+-2Cl− cotransport in aortic endothelial cells is activated by cell shrinkage, inhibited by cell swelling, and is responsible for recovery of cell volume. The role of protein phosphorylation in the regulation of cotransport was examined with two inhibitors of protein phosphatases, okadaic acid and calyculin, and a protein kinase inhibitor, K252a. Both phosphatase inhibitors stimulated cotransport in isotonic medium, with calyculin, a more potent inhibitor of protein phosphatase I, being 50-fold more potent. Neither agent stimulated cotransport in hypertonic medium. Stimulation by calyculin was immediate and was complete by 5 min, with no change in cell Na + K content, indicating that the stimulation of cotransport was not secondary to cell shrinkage. The time required for calyculin to activate cotransport was longer in swollen cells than in normal cells, indicating that the phosphorylation step is affected by cell volume. Activation of cotransport when cells in isotonic medium were placed in hypertonic medium was more rapid than the inactivation of cotransport when cells in hypertonic medium were placed in isotonic medium, which is consistent with a shrinkage-activated kinase rather than a shrinkage-inhibited phosphatase. K252a, a nonspecific protein kinase inhibitor, reduced cotransport in both isotonic and hypertonic media. The rate of inactivation was the same in either medium, indicating that dephosphorylation is not regulated by cell volume. These results demonstrate that Na+-K+-2Cl− cotransport is activated by protein phosphorylation and is inactivated by a Type I protein phosphatase. The regulation of cotransport by cell volume is due to changes in the rate of phosphorylation rather than dephosphorylation, suggesting the existence of a volume-sensitive protein kinase. Both the kinase and the phosphatase are constitutively active, perhaps to allow for rapid changes in cotransport activity.

Vasoconstrictors and nitrovasodilators reciprocally regulate the Na+-K+-2Cl−cotransporter in rat aorta

Little is known about the function and regulation of the Na+-K+-2Cl-cotransporter NKCC1 in vascular smooth muscle. The activity of NKCC1 was measured as the bumetanide-sensitive efflux of86Rb+from intact smooth muscle of the rat aorta. Hypertonic shrinkage (440 mosmol/kgH2O) rapidly doubled cotransporter activity, consistent with its volume-regulatory function. NKCC1 was also acutely activated by the vasoconstrictors ANG II (52%), phenylephrine (50%), endothelin (53%), and 30 mM KCl (54%). Both nitric oxide and nitroprusside inhibited basal NKCC1 activity (39 and 34%, respectively), and nitroprusside completely reversed the stimulation by phenylephrine. The phosphorylation of NKCC1 was increased by hypertonic shrinkage, phenylephrine, and KCl and was reduced by nitroprusside. The inhibition of NKCC1 significantly reduced the contraction of rat aorta induced by phenylephrine (63% at 10 nM, 26% at 30 nM) but not by KCl. We conclude that the Na+-K+-2Cl-cotransporter in vascular smooth muscle is reciprocally regulated by vasoconstrictors and nitrovasodilators and contributes to smooth muscle contraction, indicating that alterations in NKCC1 could influence vascular smooth muscle tone in vivo.

Glucocorticoids mediate a decrease in AVP-regulated urea transporter in diabetic rat inner medulla

Providing glucocorticoids to adrenalectomized (Adx) rats results in downregulation of the vasopressin (AVP)-regulated urea transporter (VRUT) in the renal inner medullary (IM) tip. To examine the physiological relevance of this response, we studied rats with uncontrolled diabetes mellitus induced by streptozotocin (STZ), since these rats have increased corticosterone production and urea excretion. We measured VRUT protein in extracts from the IM tip or base of pair-fed control and diabetic rats by Western analysis using an antibody to rat VRUT. In the IM tip, VRUT was significantly reduced by 39% in diabetic compared with control rats. In the IM base, there was no significant difference between diabetic and control rats. To determine whether the decrease in VRUT in the IM tip was mediated by glucocorticoids, the experiment was repeated using the following three groups of rats: 1) Adx alone, 2) Adx + STZ, and 3) Adx + STZ + replacement with a physiological dose of glucocorticoid. There was no significant difference in VRUT between Adx and Adx + STZ rats. However, VRUT was significantly reduced by 32% in the IM tip of glucocorticoid-treated Adx + STZ rats compared with control Adx + STZ rats. We conclude that glucocorticoids regulate the abundance of VRUT protein independently of insulin in diabetic rats.

Loss of N-Linked Glycosylation Reduces Urea Transporter UT-A1 Response to Vasopressin

The vasopressin-regulated urea transporter (UT)-A1 is a transmembrane protein with two glycosylated forms of 97 and 117 kDa; both are derived from a single 88-kDa core protein. However, the precise molecular sites and the function for UT-A1 N-glycosylation are not known. In this study, we compared Madin-Darby canine kidney cells stably expressing wild-type (WT) UT-A1 to Madin-Darby canine kidney cell lines stably expressing mutant UT-A1 lacking one (A1m1, A1m2) or both glycosylation sites (m1m2). Site-directed mutagenesis revealed that UT-A1 has two glycosylation sites at Asn-279 and -742. Urea flux is stimulated by 10 nm vasopressin (AVP) or 10 μm forskolin (FSK) in WT cells. In contrast, m1m2 cells have a delayed and significantly reduced maximal urea flux. A 15-min treatment with AVP and FSK significantly increased UT-A1 cell surface expression in WT but not in m1m2 cells, as measured by biotinylation. We confirmed this finding using immunostaining. Membrane fractionation of the plasma membrane, Golgi, and endoplasmic reticulum revealed that AVP or FSK treatment increases UT-A1 abundance in both Golgi and plasma membrane compartments in WT but not in m1m2 cells. Pulse-chase experiments showed that UT-A1 half-life is reduced in m1m2 cells compared with WT cells. Our results suggest that mutation of the N-linked glycosylation sites reduces urea flux by reducing UT-A1 half-life and decreasing its accumulation in the apical plasma membrane. In vivo, inner medullary collecting duct cells may regulate urea uptake by altering UT-A1 glycosylation in response to AVP stimulation.