Countercurrent Multiplication Research Articles

Oxygen delivery to the fish eye: Root effect as crucial factor for elevated retinalPO2

Although the retina has one of the highest metabolic rates among tissues, certain teleost fishes lack any vascular supply to this organ which, in combination with the overall thickness of the organ, results in extremely long diffusion distances. As the only way to compensate for these obstacles, oxygen partial pressure (PO2) in the eyes of such fish is elevated far above atmospheric values. Although not supported by any direct evidence, the enhancement of PO2 is considered to be related to the Root effect, the release upon acidification of Hb-bound O2 into physical dissolution, possibly supported by counter-current multiplication similar to the loop of Henle. The present study evaluates the magnitude of intraocular PO2 enhancement under tightly controlled physiological conditions, to directly confirm the involvement of the Root effect on intraocular PO2 in the retina of rainbow trout Oncorhynchus mykiss. Intraocular PO2 was determined with special polarographic microelectrodes inserted into the eye. PO2 profiles established in vivo by driving electrodes through the entire retina yielded average PO2 values between 10 mmHg (1.3 kPa) at the inner retinal surface and 382 mmHg (50.9 kPa) close to the outer retinal limit (Bruch's membrane). According to estimates on the basis of the diffusion distances determined from sections of the retina (approximately 436 microm at the site of PO2 measurement) and literature data on specific oxygen consumption, the in vivo determined values would be sufficient to cover the oxygen demand of the retina with some safety margin. For a clear and direct in-tissue-test as to the involvement of the Root effect, an isolated in vitro eye preparation was established in order to avoid the problem of indirect blood supply to the eye from the dorsal aorta only via the pseudobranch, a hemibranch thought to modulate blood composition before entry of the eye. Any humoral effects (e.g. catecholamines) were eliminated by perfusing isolated eyes successively with standardized red blood cell (RBC) suspensions in Ringer, using trout (with Root) and human (lacking any Root effect) RBC suspension. To optimize perfusate conditions for maximal Root effect, the Root effect of trout RBCs was determined in vitro via graded acidification of individual samples equilibrated with standardized gas mixtures. During perfusion with trout RBC, PO2 at the outer retinal limit was 99 mmHg (13.2 kPa), but fell by a factor of 3.3 upon perfusion with human RBC in spite of higher total oxygen content (TO2 2.8 for trout vs 3.9 mmol l-1 for human RBC). Upon reperfusion with trout RBC, PO2 was restored immediately to the original value. This regularly observed pattern indicated a highly significant difference (P=0.003) between perfusion with trout (with Root effect; high retinal PO2) and perfusion with human (no Root effect; low retinal PO2) RBC suspension, thus clearly demonstrating that the Root effect is directly involved and a crucial prerequisite for the enhancement of PO2 in the retina of the teleost eye.

Molecular Mechanisms of Impaired Urinary Concentrating Ability in Glucocorticoid-Deficient Rats

The purpose of this study was to examine urinary concentrating ability and protein expression of renal aquaporins and ion transporters in glucocorticoid-deficient (GD) rats in response to water deprivation as compared with control rats. Rats underwent bilateral adrenalectomies, followed only by aldosterone replacement (GD) or both aldosterone and dexamethasone replacement (control). As compared with control rats, the GD rats demonstrated a decrease in cardiac output and mean arterial pressure. In response to 36-h water deprivation, GD rats demonstrated significantly greater urine flow rate and decreased urine osmolality as compared with control rats at comparable serum osmolality and plasma vasopressin concentrations. The initiator of the countercurrent concentrating mechanism, the sodium-potassium-2 chloride co-transporter, was significantly decreased, as was the medullary osmolality in the GD rats versus control rats. There was also a decrease in inner medulla aquaporin-2 (AQP2) and urea transporter A1 (UT-A1) in GD rats as compared with control rats. There was a decrease in outer medulla Gsalpha protein, an important factor in vasopressin-mediated regulation of AQP2. Immunohistochemistry studies confirmed the decreased expression of AQP2 and UT-A1 in kidneys of GD rats as compared with control. In summary, impairment in the urinary concentrating mechanism was documented in GD rats in association with impaired countercurrent multiplication, diminished osmotic equilibration via AQP2, and diminished urea equilibration via UT-A1. These events occurred primarily in the relatively oxygen-deficient medulla and may have been initiated, at least in part, by the decrease in mean arterial pressure and thus renal perfusion pressure in this area of the kidney.

Altered expression profile of transporters in the inner medullary collecting duct of aquaporin-1 knockout mice

Aquaporin-1 is the major protein responsible for transport of water across the epithelia of the proximal tubule and thin descending limbs. Rapid water efflux across the thin descending limb is required for the normal function of the countercurrent multiplier mechanism. Therefore, urinary concentrating capacity is severely impaired in aquaporin-1 knockout (AQP1 -/-) mice. Here, we have investigated the long-term consequences of deletion of the AQP1 gene product by profiling abundance changes in transporters expressed in the inner medullas of AQP1 (-/-) mice vs. heterozygotes [AQP1 (+/-)], which have a normal concentrating capacity. Semiquantitative immunoblotting demonstrated marked suppression of two proteins strongly expressed in the inner medullary collecting duct (IMCD): UT-A1 (a urea transporter) and AQP4 (a basolateral water channel). Furthermore, the urea permeability of the IMCD was significantly reduced in AQP1 (-/-) mice. In contrast, there was increased expression of three proteins normally expressed at higher levels in the cortical collecting duct (CCD) than in IMCD: AQP3 (another basolateral water channel) and the epithelial sodium channel subunits beta-ENaC and gamma-ENaC. Changes in expression of these proteins were confirmed by immunocytochemistry. Messenger RNA profiling (real-time RT-PCR) revealed changes in UT-A1, beta-ENaC, gamma-ENaC, and AQP3 transcript abundance that paralleled the changes in protein abundance. Thus, from the perspective of transport proteins, the IMCDs of AQP1 (-/-) mice have a significantly altered phenotype. To address whether these changes are specific to AQP1 (-/-) mice, we profiled IMCD transporter expression in a second knockout model manifesting a concentrating defect, that of ClC-nK1, a chloride channel in the ascending thin limb important for urinary concentration. As in the AQP1 knockout mice, ClC-nK1 (-/-) mice showed decreased expression of UT-A1 and increased expression of beta-ENaC and gamma-ENaC vs. WT controls. In conclusion, the expression profile of IMCD transporters is markedly altered in AQP1 -/- mice and this manifestation is related to the associated concentrating defect.

Differential Effects of Hyperosmolality on Na-K-ATPase and Vasopressin-Dependent cAMP Generation in the Medullary Thick Ascending Limb and Outer Medullary Collecting Duct

Hyperosmolality in the renal medullary interstitium is generated by the renal countercurrent multiplication system, in which the medullary thick ascending limb (MAL) and the outer medullary collecting duct (OMCD) primarily participate. Since arginine vasopressin (AVP) regulates Na-K-ATPase activity directly via protein kinase A and indirectly via hyperosmolality, we investigated the acute and chronic effects of hyperosmolality on Na-K-ATPase and AVP-dependent cAMP generation in the MAL and OMCD. Microdissected MAL and OMCD from control and dehydrated rats were used for the measurement of Na-K-ATPase activity, mRNA expression of alpha-1, beta-1, and beta-2 subunits of Na-K-ATPase, and AVP-dependent cAMP generation. Na-K-ATPase activity in the MAL from dehydrated rats, as measured in isotonic medium, was higher than that of control rats. Moreover, incubation of samples in hypertonic medium (490 mOsm/kg H2O) further increased Na-K-ATPase activity. Dehydration increased alpha-1, beta-1, and beta-2 mRNA expression in the MAL without changing that in the OMCD. Western blot analysis revealed that in the outer medulla, the expression of beta-1, but not that of alpha-1 or beta-2, was stimulated by dehydration. Incubation of MAL or OMCD in hypertonic medium increased AVP-dependent cAMP generation. Higher levels of AVP-dependent cAMP were generated in the MAL from dehydrated rats than that of controls, although incubation in hypertonic medium did not lead to additional increases in AVP-dependent cAMP accumulation. In contrast, AVP-dependent cAMP generation in the OMCD was stimulated by dehydration, and was further stimulated by incubation in hypertonic medium. These findings demonstrate that Na-K-ATPase is upregulated short- and long-term hyperosmolality in the MAL, but not in OMCD.

Renal medullary gene expression in aquaporin-1 null mice

Mice that lack the aquaporin-1 gene (AQP1) lack a functional countercurrent multiplier mechanism, fail to concentrate the inner medullary (IM) interstitium, and present with a urinary concentrating defect. In this study, we use DNA microarrays to identify the gene expression profile of the IM of AQP1 null mice and corresponding changes in gene expression resulting from a loss of a hypertonic medullary interstitium. An ANOVA analysis model, CARMA, was used to isolate the knockout effect while taking into account experimental variability associated with microarray studies. In this study 5,701 genes of the possible approximately 12,000 genes on the array were included in the ANOVA; 531 genes were identified as demonstrating a >1.5-fold up- or downregulation between the wild-type and knockout groups. We randomly selected 35 genes for confirmation by real-time PCR, and 29 of the 35 genes were confirmed using this method. The overall pattern of gene expression in the AQP1 null mice was one of downregulation compared with gene expression in the renal medullas of the wild-type mice. Heat shock proteins 105 and 94, aldose reductase, adenylate kinase 2, aldolase B, aldehyde reductase 6, and p8 were decreased in the AQP1 null mice. Carboxylesterase 3, matrilin 2, lipocalin 2, and transforming growth factor-alpha were increased in IM of AQP1 null mice. In addition, we observed a loss of vasopressin type 2 receptor mRNA expression in renal medullas of the AQP1 null mice. Thus the loss of the hyperosmotic renal interstitium, due to a loss of the concentrating mechanism, drastically altered not only the phenotype of these animals but also their renal medullary gene expression profile.

Renal Sodium Handling for Body Fluid Maintenance and Blood Pressure Regulation

AbstractFor Abstract see ChemInform Abstract in Full Text.

Molecular and functional characterization of a vasotocin-sensitive aquaporin water channel in quail kidney.

Both mammals and birds can concentrate urine hyperosmotic to plasma via a countercurrent multiplier mechanism, although evolutionary lines leading to mammals and birds diverged at an early stage of tetrapod evolution. We reported earlier (Nishimura H, Koseki C, and Patel TB. Am J Physiol Regul Integr Comp Physiol 271: R1535-R1543, 1996) that arginine vasotocin (AVT; avian antidiuretic hormone) increases diffusional water permeability in the isolated, perfused medullary collecting duct (CD) of the quail kidney. In the present study, we have identified an aquaporin (AQP) 2 homolog water channel in the medullary cones of Japanese quail, Coturnix coturnix (qAQP2), by RT-PCR-based cloning techniques. A full-length cDNA contains an 822-bp open reading frame that encodes a 274-amino acid sequence with 75.5% identity to rat AQP2. The qAQP2 has six transmembrane domains, two asparagine-proline-alanine (NPA) sequences, and putative N-glycosylation (asparagine-124) and phosphorylation sites (serine-257) for cAMP-dependent protein kinase. qAQP2 is expressed in the membrane of Xenopus laevis oocytes and significantly increased its osmotic water permeability (P(f)), inhibitable (P < 0.01) by mercury chloride. qAQP2 mRNA (RT-PCR) was detected in the kidney; medullary mRNA levels were higher than cortical levels. qAQP2 protein that binds to rabbit anti-rat AQP2 antibody is present in the apical/subapical regions of both cortical and medullary CDs from normally hydrated quail, and the intensity of staining increased only in the medullary CDs after water deprivation or AVT treatment. The relative density of the approximately 29-kDa protein band detected by immunoblot from the medullary cones was modestly higher in water-deprived/AVT-treated quail. The results suggest that 1) medullary CDs of quail kidneys express a mercury-sensitive functioning qAQP2 water channel, and 2) qAQP2 is at least partly regulated by an AVT-dependent mechanism. This is the first clear identification of AQP2 homolog in nonmammalian vertebrates.

Renal sodium handling for body fluid maintenance and blood pressure regulation.

Renal sodium handling is an essential physiologic function in mammal for body fluid maintenance and blood pressure regulation. Recent advances in molecular biology have led to the identification of kidney-specific sodium transporters in the renal tubule, thereby supplying vast information for renal physiology as well as systemic physiology. Renal urinary concentration for body fluid maintenance is accomplished by counter current multiplication in the distal tubule. Sodium transport in the thick ascending limb of Henle (TAL) is the initial process of this system. We have demonstrated that renal urinary concentration is regulated in part by the expression of the Na(+)-K(+)-2Cl(-) co-transporter (BSC1) in TAL, by showing two mechanisms of BSC1 expression: pitressin vasopressin (AVP)-dependent and AVP-independent mechanisms. Two additional findings, namely, a lack of the ability to increase BSC1 expression leads to urinary concentrating defect and an enhanced BSC1 expression underlies the edema-forming condition, confirm the close association between sodium handling in TAL and body fluid accumulation. The lines of evidence from our genetic studies of the general Japanese population suggest the importance of mendelian hypertension genes in the genetic investigation of essential hypertension. Because those genes directly or indirectly regulate sodium transport by the Na-Cl co-transporter or the epithelial sodium channel in the distal convoluted tubule to the collecting duct (distal tubular segments after TAL), sodium handling in this part of the renal tubule may be, at least in part, involved in blood pressure regulation. The unveiling of such physiologic roles of sodium handling based on the sodium transporters or on the tubular segments may lead to a better understanding of systemic physiology as well as to the development of novel therapy for body fluid or blood pressure disorders.

The fall and rise of active chloride transport: implications for regulation of intraocular pressure.

Early study of transepithelial salt transfer focused on Cl(-) and not Na(+), partly because Cl(-) was readily measureable. The advent of flame photometry and tracer techniques brought Na(+) to the fore, especially since short-circuited frog skin (Rana temporaria) produces baseline net movement of Na(+) and not of Cl(-). Zadunaisky was among the first to describe what is currently termed secondary active Cl(-) transport, helping stimulate interest in Cl(-) handling by other tissues, notably the thick ascending limb of the loop of Henle important in renal counter-current multiplication. More recently, molecules responsible for electroneutral and electrogenic Cl(-) transfer have been cloned, and specific diseases resulting from their faulty expression have been identified. The clinical importance of transepithelial Cl(-) transfer is illustrated by studies of aqueous humor formation by the eye's bilayered ciliary epithelium. NaCl is taken up from the stroma by the pigmented ciliary epithelial (PE) layer, diffuses through gap junctions into the nonpigmented ciliary epithelial (NPE) layer, and is released into the aqueous humor largely through Na(+) pumps and Cl(-) channels. ATP released by NPE cells can be ecto-enzymatically metabolized to adenosine. Adenosine can mediate paracrine/autocrine stimulation of Cl(-) channels and aqueous humor secretion by occupying A(3) adenosine receptors (ARs). A(3)AR agonists indeed elevate, and A(3)AR antagonists lower, intraocular pressure (IOP) in wild-type mice. A(3)AR knockout mice have low IOP and their responses to A(3)AR agonists and antagonists are blunted; this suggests that reducing Cl(-)-channel activity with A(3)AR antagonists may provide a novel approach for treating glaucoma.

Vasopressin-independent renal urinary concentration: Increased rBSC1 and enhanced countercurrent multiplication

A close association between the expression of the sodium transporter, rat bumetanide sensitive cotransporter (rBSC), in thick ascending limb of Henle and urinary concentration has been reported. However, direct evidence for this association and the mechanism of rBSC1 expression are still to be elucidated. Brattleboro (BB) rats weighing approximately 200 g were dehydrated by water restriction for 4 hours, which induced around a 5% body weight reduction. Although plasma arginine vasopressin (AVP) was undetectable even after the water restriction, BB rats concentrated urine from 182 +/- 23 (mean +/- SD) at baseline to 404 +/- 65 mOsm/kg. H2O. Urinary volume was reduced from 5.8 +/- 1.8 to 1.4 +/- 0.6 mL/h. This treatment significantly increased sodium and urea accumulation in the renal medulla and reduced urinary sodium excretion. rBSC1 signals for both mRNA and protein were increased in dehydrated rats, although aquaporin type 2 (AQP2) expression was not enhanced in dehydrated BB rats. Subcutaneous infusion of desmopressin acetate (DDAVP) intensified rBSC1 signals of BB rats more than those in dehydrated condition. Dehydration increased rBSC1 expression and enhanced countercurrent multiplication even in AVP deficiency. These results supply strong evidence for the association between rBSC1 expression and urinary concentration, and indicate the presence of an AVP-independent mechanism for urine concentration.

Urinary concentrating defect in hypothyroid rats: role of sodium, potassium, 2-chloride co-transporter, and aquaporins.

Hypothyroidism is associated with impaired urinary concentrating ability in humans and animals. The purpose of this study was to examine protein expression of renal sodium chloride and urea transporters and aquaporins in hypothyroid rats (HT) with diminished urinary concentration as compared with euthyroid controls (CTL) and hypothyroid rats replaced with L-thyroxine (HT+T). Hypothyroidism was induced by aminotriazole administration. Body weight, water intake, urine output, solute and urea excretion, serum and urine osmolality, serum creatinine, 24-h creatinine clearance, and fractional excretion of sodium were comparable among the three groups. However, with 36 h of water deprivation, HT rats demonstrated significantly greater urine flow rates and decreased urine and medullary osmolality as compared with CTL and HT+T rats at comparable plasma vasopressin concentrations. Western blot analyses revealed decreased renal protein abundance of transporters, including Na-K-2Cl, Na-K-ATPase, and NHE3, in HT rats as compared with CTL and HT+T rats. Protein abundance of renal AQP1 and urea transporters UTA(1) and UTA(2) did not differ significantly among study groups. There was however a significant decrease in protein abundance of AQP2, AQP3, and AQP4 in HT rats as compared with CTL and HT+T rats. These findings demonstrate a decrease in the medullary osmotic gradient secondary to impaired countercurrent multiplication and downregulation of aquaporins 2, 3, and 4 as contributors to the urinary concentrating defect in the hypothyroid rat.

The osmotic gradient in kidney medulla: a retold story.

This article is an attempt to simplify lecturing about the osmotic gradient in the kidney medulla. In the model presented, the kidneys are described as a limited space with a positive interstitial hydrostatic pressure. Traffic of water, sodium, and urea is described in levels (or horizons) of different osmolarity, governed by osmotic forces and positive interstitial pressure. In this way, actions of the countercurrent multiplier in nephron tubules and of the countercurrent exchanger in vasa recta are integrated in each horizon. We hope that this approach can help students to better accept conventional presentations in their textbooks.

Water Absorption Enhances the Uptake of Mannitol and Decreases Cr-EDTA/Mannitol Permeability Ratios in Cat Small Intestine In Situ

Background: Recently, we hypothesized that mannitol absorption in human intestinal permeability tests is a reflection of small intestinal water absorption and is dependent mainly on the efficiency of the countercurrent multiplier in the villi. This may affect the outcome of clinical double-sugar permeability tests. We tested the hypothesis in cats, another species with an efficient countercurrent multiplier. Methods: The lumen-to-tissue transport of [ 14 C]mannitol and [ 51 Cr]EDTA was studied in in situ perfused jejunum of eight anaesthetized cats using four isotonic perfusion solutions with varying sodium and glucose content. The transport of water was monitored, and the absorption rate of the probes was calculated by their disappearance from the perfusate. Results: There was a significant positive correlation between water absorption and [ 14 C]mannitol clearance from the different perfusates ( r = 0.99; P < 0.01), whereas this correlation was absent for [ 51 Cr]EDTA clearance ( r = 0.05; P = 0.95). There was also a significant negative correlation between water absorption and [ 51 Cr]EDTA/[ 14 C]mannitol clearance ratios ( r = 0.98; P < 0.02). Conclusions: The results show a prominent effect of water absorption on mannitol uptake through pores which, also during glucose transport, exclude Cr-EDTA. The difference in water absorption from the solutions used in cat small intestine is dependent on the effectiveness of the countercurrent multiplier; we conclude that the capability of this mechanism influences mannitol absorption in vivo. Qualitatively comparable results were obtained using oral test solutions with varying NaCl and glucose concentrations in human volunteers. We propose that the functioning of the countercurrent multiplier is essential for the interpretation of double-sugar tests in clinical studies.

The Kidney at a Glance

What a marvellous book: would that such had been available to me as a medical student nearly 40 years ago when I grappled semicomprehendingly with types I and II nephritis and the countercurrent multiplier system. The kidney is, of course, the most sophisticated and fascinating of organs—the heart a mere pump; the brain an electrical junction box; the lungs crude bellows—and those of us who treat its malfunctions sit above such lowly specialties. The kidney protects us from dehydration and biochemical disequilibration; filters metabolic and immunological garbage (recycling where possible); allows us to stand erect without our blood pressure and bodies tumbling; keeps our blood red and our bones strong; and limits the calamitous results of doctors' tendency to prescribe drugs and patients' enthusiasm for taking them. Small wonder that renal failure was the first replacement endeavour: and how spectacularly successful it has been. By now you will need no persuading of the imperative for a sound undergraduate renal textbook, and The Kidney at a Glance, from the youthful O'Callaghan and the seasoned Brenner, offers the necessary blend of immediacy and balance. The forty-plus chapters give generous glances (lingering looks actually) at the topics mentioned above, and many more, each in a couple of crisp pages containing well labelled diagrams and easy-to-follow text, broken up with bullet points and other modern publishing techniques to make the information stick. Also, a website is available for help and to test one's newly minted knowledge against multiple choice questions. (A set of questions is provided for reviewers of the book, and on this, luckily for the publisher, I got full marks.) There is a formidable index. Any postgraduate examiner would obtain satisfaction from a candidate possessing half the information in this book, and it would be helpful to mature specialists such as myself needing a shot of revision before delivering a lecture on, say, tubular physiology. And yet, and yet... Where is the passion? I have been a privileged navigator to scores of patients and relatives steering through the arduous journey of renal disease. Many have symptoms which I have only been able to palliate and all the molecular biology in the world is not going to help end-of-the-road patients and relatives wracked by the decision whether to have one last crack at interventionist medicine. This is the warp and woof of bedside renal medicine which few textbooks come near to conveying and which gives nephrologists the greatest rewards—knowledge of having been of personal assistance. And where is the context? How will an undergraduate fresh to renal medicine know that renal stones are more common in children in Karachi than Kidderminster and that unexplained and life-demeaning urinary frequency and urgency are more common than nephrotic syndrome? Presumably the educationalists, assembling the joined-up medical curriculum, will ensure these are covered in the ethics and communication skills and epidemiology courses, but I wonder whether students will make the necessary links with clinical practice. Academic nephrologists neglect or delegate these wider aspects of our blessed specialty at its peril; the young watch and mark. And now an admission. My daughter cramming for part 1 MRCP(UK) spotted the review copy on my desk, skimmed it through, declared it ‘cool’ and filched it. Perhaps her judgment is the one to be heeded.

Water Absorption Enhances the Uptake of Mannitol and Decreases Cr-EDTA/Mannitol Permeability Ratios in Cat Small Intestine In Situ

Background: Recently, we hypothesized that mannitol absorption in human intestinal permeability tests is a reflection of small intestinal water absorption and is dependent mainly on the efficiency of the countercurrent multiplier in the villi. This may affect the outcome of clinical double-sugar permeability tests. We tested the hypothesis in cats, another species with an efficient countercurrent multiplier. Methods: The lumen-to-tissue transport of [ 14 C]mannitol and [ 51 Cr]EDTA was studied in in situ perfused jejunum of eight anaesthetized cats using four isotonic perfusion solutions with varying sodium and glucose content. The transport of water was monitored, and the absorption rate of the probes was calculated by their disappearance from the perfusate. Results: There was a significant positive correlation between water absorption and [ 14 C]mannitol clearance from the different perfusates ( r = 0.99; P < 0.01), whereas this correlation was absent for [ 51 Cr]EDTA clearance ( r = 0.05; P = 0.95). There was also a significant negative correlation between water absorption and [ 51 Cr]EDTA/[ 14 C]mannitol clearance ratios ( r = 0.98; P < 0.02). Conclusions: The results show a prominent effect of water absorption on mannitol uptake through pores which, also during glucose transport, exclude Cr-EDTA. The difference in water absorption from the solutions used in cat small intestine is dependent on the effectiveness of the countercurrent multiplier; we conclude that the capability of this mechanism influences mannitol absorption in vivo. Qualitatively comparable results were obtained using oral test solutions with varying NaCl and glucose concentrations in human volunteers. We propose that the functioning of the countercurrent multiplier is essential for the interpretation of double-sugar tests in clinical studies.

Aquaporins: Roles in Renal Function and Peritoneal Dialysis

Aquaporin (AQP) water channels are important in the function of the kidney. Constitutively expressed AQP1 in the proximal tubule and descending limb is important in normal fluid absorption and in the counter-current multiplication system. The vasopressin-regulated shuttling of AQP2 is essential in antidiuresis and the regulation of water balance. Genetic damage to AQPs, or pathological changes in expression or function, impair renal water handling. The most striking examples of this involve disruption of AQP2 function, which can result in profound nephrogenic diabetes insipidus. Aquaporin 1 is present in capillaries and venules and appears to be important in peritoneal dialysis, where it appears to represent the "ultrasmall pores" of the three-pore model. Decreased expression or function of AQP1 may be responsible for some cases of ultrafiltration failure, but further evidence will be required to establish whether this is the case.

A brief history of theories concerning the mammalian urine concentrating mechanism.

The mechanism by which the mammalian kidney creates the osmotic gradient necessary for urine concentration remains an open question. We present a brief survey of the give-and-take between theory and experiment on this question over the last half century. We start with the introduction of the countercurrent multiplier paradigm in 1951. We finish with a description of a recent suggestion that the explanation for the enigmatic inner medullary osmotic gradient may reside in the very metabolism of the inner medullary cells, which are required by the region's hypoxia to obtain their ATP largely from anaerobic glycolysis and which thus, by the same token, furnish net osmoles to the medullary interstitium by converting glucose to lactate.

Regulation of potassium channel Kir 1.1 (ROMK) abundance in the thick ascending limb of Henle's loop.

The renal outer medullary potassium channel (ROMK) of the thick ascending limb (TAL) is a critical component of the counter-current multiplication mechanism. In this study, two new antibodies raised to ROMK were used to investigate changes in the renal abundance of ROMK with treatments known to strongly promote TAL function. These antibodies specifically recognized protein of the predicted size of 45 kD in immunoblots of rat kidney or COS cells transfected with ROMK cDNA. Infusion of 1-deamino-(8-D-arginine)-vasopressin (dDAVP), a vasopressin V2 receptor-selective agonist, for 7 d into Brattleboro rats resulted in dramatic increases in apical membrane labeling of ROMK in the TAL of dDAVP-treated rats, as assessed by immunocytochemical analyses. Using immunoblotting, a more than threefold increase in immunoreactive ROMK levels was observed in the outer medulla after dDAVP infusion. Restriction of water intake to increase vasopressin levels also significantly increased TAL ROMK immunolabeling and abundance in immunoblots. In addition, dietary Na(+) levels were varied to determine whether ROMK abundance was also affected under other conditions known to alter TAL transport. Rats fed higher levels of sodium, as either NaCl or NaHCO(3) (8 mEq/250 g body wt per d), exhibited significantly increased density of the 45-kD band, compared with the respective control animals. Moreover, in rats fed a low-NaCl diet (0.25 mEq/250 g body wt per d), a 50% decrease in band density for the 45-kD band was observed (relative to control rats fed 2.75 mEq/250 g body wt per d of NaCl). These results demonstrate that long-term adaptive changes in ROMK abundance occur in the TAL with stimuli that enhance transport by this segment.

Mathematical model of an avian urine concentrating mechanism.

A mathematical model was used to investigate how concentrated urine is produced within the medullary cones of the quail kidney. Model simulations were consistent with a concentrating mechanism based on single-solute countercurrent multiplication and on NaCl cycling from ascending to descending limbs of loops of Henle. The model predicted a urine-to-plasma (U/P) osmolality ratio of approximately 2.26, a value consistent with maximum avian U/P osmolality ratios. Active NaCl transport from descending limb prebend thick segments contributed 70% of concentrating capability. NaCl entry and water extraction provided 80 and 20%, respectively, of the concentrating effect in descending limb flow. Parameter studies indicated that urine osmolality is sensitive to the rate of fluid entry into descending limbs and collecting ducts at the cone base. Parameter studies also indicated that the energetic cost of concentrating urine is sensitive to loop of Henle population as a function of medullary depth: as the fraction of loops reaching the cone tip increased above anatomic values, urine osmolality increased only marginally, and, ultimately, urine osmolality decreased.

Impact of K+ homeostasis on net acid secretion in rat terminal inner medullary collecting duct: Role of the Na+,K-ATPase

For the past 50 years, the mechanism of ammonium (NH4+ ) transport along the collecting duct has been thought to occur through active H+ secetion in parallel with the nonionic diffusion of ammonia (NH3 ). This model is supported by two basic experimental observations. First, NH4+ secretion generally correlates with the NH3 concentration gradient between the interstitium and the collecting duct lumen. This NH3 gradient is generated through both luminal acidification, which reduces luminal NH3 concentration, and through countercurrent multiplication, which increases interstitial NH3 concentration. The result is secretion of NH3 into the collecting duct lumen down its concentration gradient. Second, because NH4+ permeability is low relative to that of NH3 , there is significant secretion of NH3 into the collecting duct lumen with minimal back-diffusion of NH4+ . However, our laboratory, as well as others, has shown that this model is an oversimplification of the mechanism of NH4+ transport along the collecting duct. NH4+ is transported through a variety of K+ transport pathways including Na,K-ATPase. K+ and NH4+ compete for a common extracellular binding site on Na,K-ATPase. During hypokalemia, interstitial K+ concentration is reduced, which augments NH4+ uptake by the Na+ pump. In K+ restriction, Na,K-ATPase–mediated NH4+ uptake provides an important source of H+ for net acid secretion and for the titration of luminal buffers in the terminal inner medullary collecting duct. This pathway contributes to the increase in NH4+ excretion and metabolic alkalosis observed during hypokalemia.