Jeff M. Sands

Apical extracellular calcium/polyvalent cation-sensing receptor regulates vasopressin-elicited water permeability in rat kidney inner medullary collecting duct.

During antidiuresis, increases in vasopressin (AVP)-elicited osmotic water permeability in the terminal inner medullary collecting duct (tIMCD) raise luminal calcium concentrations to levels (> or = 5 mM) above those associated with the formation of calcium-containing precipitates in the urine. Calcium/polycation receptor proteins (CaRs) enable cells in the parathyroid gland and kidney thick ascending limb of Henle to sense and respond to alterations in serum calcium. We now report the presence of an apical CaR in rat kidney tIMCD that specifically reduces AVP-elicited osmotic water permeability when luminal calcium rises. Purified tIMCD apical membrane endosomes contain both the AVP-elicited water channel, aquaporin 2, and a CaR. In addition, aquaporin 2-containing endosomes also possess stimulatory (G(alpha q)/G(alpha 11) and inhibitory (G(alpha i1, 2, and 3)) GTP binding proteins reported previously to interact with CaRs as well as two specific isoforms (delta and zeta) of protein kinase C. Immunocytochemistry using anti-CaR antiserum reveals the presence of CaR protein in both rat and human collecting ducts. Together, these data provide support for a unique tIMCD apical membrane signaling mechanism linking calcium and water metabolism. Abnormalities in this mechanism could potentially play a role in the pathogenesis of renal stone formation.

Vasopressin-elicited water and urea permeabilities are altered in IMCD in hypercalcemic rats

To investigate how hypercalcemia blunts renal concentrating ability, alterations in basal and arginine vasopressin (AVP)-elicited osmotic water ( P f) and urea ( P urea) permeabilities were measured in isolated perfused terminal inner medullary collecting ducts (IMCD) from control and chronically hypercalcemic rats after dihydrotachysterol (DHT) (M. Levi, L. Peterson, and T. Berl. Kidney Int. 23: 489-497, 1983) treatment. The IMCD P f of DHT-treated rats did not increase significantly after AVP and was accompanied by a significant 87 ± 4% reduction in aquaporin-2 (AQP-2) protein but not mRNA. In contrast, both basal and AVP-elicited IMCD P urea from DHT rats were significantly increased and accompanied by a significant 41 ± 11% increase in AVP-regulated urea transporter protein content. Immunoblotting with anti-calcium/polyvalent cation-sensing receptor protein (CaR) antiserum revealed specific alterations in CaR bands in endosomes purified from the apical membranes of inner medulla of DHT rats. These data are the first detailed analyses of hypercalcemia-induced alterations in AVP-regulated permeabilities and membrane transporters in IMCD. We conclude that selective alterations in IMCD transport occur in hypercalcemia, permitting the body to dispose of excess calcium without forming calcium-containing renal stones.

Atrial natriuretic factor inhibits vasopressin-stimulated osmotic water permeability in rat inner medullary collecting duct.

The inner medullary collecting duct (IMCD) has been proposed to be a site of atrial natriuretic factor (ANF) action. We carried out experiments in isolated perfused terminal IMCDs to determine whether ANF (rat ANF 1-28) affects either osmotic water permeability (Pf) or urea permeability. In the presence of a submaximally stimulating concentration of vasopressin (10(-11) M), ANF (100 nM) significantly reduced Pf by an average of 46%. Lower concentrations of ANF also significantly inhibited vasopressin-stimulated Pf by the following percentages: 0.01 nM ANF, 18%; 0.1 nM, 46%; 1 nM, 48%. Addition of exogenous cyclic GMP (0.1 mM) mimicked the effect of ANF, decreasing Pf by an average of 48%. ANF also inhibited cyclic AMP-stimulated Pf by an average of 31%. ANF did not affect urea permeability, nor did it alter vasopressin-stimulated cyclic AMP accumulation. We conclude that ANF at physiological concentrations causes a large inhibition of vasopressin-stimulated Pf in the rat terminal IMCD, and that cyclic GMP is the second messenger mediating the effect. ANF appears to act at a site distal to cyclic AMP generation in the chain of events linking vasopressin receptor binding to an increase in osmotic water permeability.

The Physiology of Urinary Concentration: An Update

The renal medulla produces concentrated urine through the generation of an osmotic gradient extending from the cortico-medullary boundary to the inner medullary tip. This gradient is generated in the outer medulla by the countercurrent multiplication of a comparatively small transepithelial difference in osmotic pressure. This small difference, called a single effect, arises from active NaCl reabsorption from thick ascending limbs, which dilutes ascending limb flow relative to flow in vessels and other tubules. In the inner medulla, the gradient may also be generated by the countercurrent multiplication of a single effect, but the single effect has not been definitively identified. There have been important recent advances in our understanding of key components of the urine concentrating mechanism. In particular, the identification and localization of key transport proteins for water, urea, and sodium, the elucidation of the role and regulation of osmoprotective osmolytes, better resolution of the anatomical relationships in the medulla, and improvements in mathematic modeling of the urine concentrating mechanism. Continued experimental investigation of transepithelial transport and its regulation, both in normal animals and in knock-out mice, and incorporation of the resulting information into mathematic simulations, may help to more fully elucidate the inner medullary urine concentrating mechanism.

Renal urea transporters.

Purpose of reviewUrea is transported across the kidney inner medullary collecting duct by urea-transporter proteins. Two urea-transporter genes have been cloned from humans and rodents: the UT-A (Slc14A2) gene encodes five protein and eight cDNA isoforms; the UT-B (Slc14A1) gene encodes a single isoform. In the past year, significant progress has been made in understanding the regulation of urea-transporter protein abundance in kidney, studies of genetically engineered mice that lack a urea transporter, identification of urea transporters outside of the kidney, cloning of urea transporters in nonmammalian species, and active urea transport in microorganisms. Recent findingsUT-A1 protein abundance is increased by 12 days of vasopressin, but not by 5 days. Analysis of the UT-A1 promoter suggests that vasopressin increases UT-A1 indirectly following a direct effect to increase the transcription of other genes, such as the Na+-K+-2Cl− cotransporter NKCC2/BSC1 and the aquaporin (AQP) 2 water channel, that begin to increase inner medullary osmolality. UT-A1 protein abundance is also increased by adrenalectomy, and is decreased by glucocorticoids or mineralocorticoids. However, each hormone works through its own receptor. Knockout mice that lack UT-A1 and UT-A3, or lack UT-B, have a urine-concentrating defect and a decrease in inner medullary interstitial urea content. SummaryUrea transporters play a critical role in the urine-concentrating mechanism. Their abundance is regulated by vasopressin, glucocorticoids, and mineralocorticoids. These regulatory mechanisms may be important in disease states such as diabetes because changes in urea-transporter abundance in diabetic rats require glucocorticoids and vasopressin.

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.

Molecular Mechanisms of Urea Transport

Physiologic data provided evidence for specific urea transporter proteins in red blood cells and kidney inner medulla. During the past decade, molecular approaches resulted in the cloning of several urea transporter cDNA isoforms derived from two gene families: UT-A and UT-B. Polyclonal antibodies were generated to the cloned urea transporter proteins, and their use in integrative animal studies resulted in several novel findings, including: (1) UT-B is the Kidd blood group antigen; (2) UT-B is also expressed in many non-renal tissues and endothelial cells; (3) vasopressin increases UT-A1 phosphorylation in rat inner medullary collecting duct; (4) the surprising finding that UT-A1 protein abundance and urea transport are increased in the inner medulla during conditions in which urine concentrating ability is reduced; and (5) UT-A protein abundance is increased in uremia in both liver and heart. This review will summarize the knowledge gained from studying molecular mechanisms of urea transport and from integrative studies into urea transporter protein regulation.

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.

Dietary Sodium Intake in Heart Failure

Dietary sodium restriction is arguably the most frequent self-care behavior recommended to patients with heart failure (HF)1,2 and is endorsed by all HF guidelines.2–10 However, the data on which this recommendation is drawn are modest, and the limited trials conducted have produced inconsistent findings. Americans consume ≈3700 mg sodium daily,11 whereas the US Department of Agriculture and the Department of Health and Human Services recommend 2300 mg daily intake for the general population, with a stricter recommendation of 1500 mg/d for those >50 years of age, blacks, or individuals with hypertension, diabetes mellitus, or chronic kidney disease.12 According to a recent report from the National Health and Nutrition Examination Survey, although 47.6% of persons aged ≥2 years meet the criteria to limit daily sodium intake to 1500 mg, the usual intake for 98.6% of those persons was >1500 mg; in 88.2% of the remaining population, daily intake was greater than the recommended <2300 mg.13 The American Heart Association now recommends sodium intake of 1500 mg/d for all Americans,14 similar to the recommendation by the Institute of Medicine.15 Interestingly, and paradoxically, the suggested 1500 mg daily sodium intake for the general population is less than the limit proposed for HF patients by most guidelines, which appears as a contradiction. Whether this contradiction suggests inconsistent policy or a limited understanding of sodium homeostasis in the HF versus non-HF state is debatable. Sodium homeostasis physiology is altered in HF as opposed to healthy individuals and those with hypertension, and may partially explain these incongruous recommendations. This review summarizes the studies assessing the effects of sodium restriction in HF, highlighting knowledge gaps and future directions. Excessive sodium intake is associated with fluid retention. Therefore, all HF management guidelines recommend sodium restriction. In 2005, …