Aurélie Edwards

Requirement of aquaporin-1 for NaCl-driven water transport across descending vasa recta

Deletion of AQP1 in mice results in diminished urinary concentrating ability, possibly related to reduced NaCl- and urea gradient-driven water transport across the outer medullary descending vasa recta (OMDVR). To quantify the role of AQP1 in OMDVR water transport, we measured osmotically driven water permeability in vitro in microperfused OMDVR from wild-type, AQP1 heterozygous, and AQP1 knockout mice. OMDVR diameters in AQP1(-/-) mice were 1.9-fold greater than in AQP1(+/+) mice. Osmotic water permeability (P(f)) in response to a 200 mM NaCl gradient (bath > lumen) was reduced about 2-fold in AQP1(+/-) mice and by more than 50-fold in AQP1(-/-) mice. P(f) increased from 1015 to 2527 microm/s in AQP1(+/+) mice and from 22 to 1104 microm/s in AQP1(-/-) mice when a raffinose rather than an NaCl gradient was used. This information, together with p-chloromercuribenzenesulfonate inhibition measurements, suggests that nearly all NaCl-driven water transport occurs by a transcellular route through AQP1, whereas raffinose-driven water transport also involves a parallel, AQP1-independent, mercurial-insensitive pathway. Interestingly, urea was also able to drive water movement across the AQP1-independent pathway. Diffusional permeabilities to small hydrophilic solutes were comparable in AQP1(+/+) and AQP1(-/-) mice but higher than those previously measured in rats. In a mathematical model of the medullary microcirculation, deletion of AQP1 resulted in diminished concentrating ability due to enhancement of medullary blood flow, partially accounting for the observed urine-concentrating defect.

A mathematical model of O2 transport in the rat outer medulla. I. Model formulation and baseline results

The mammalian kidney is particularly vulnerable to hypoperfusion, because the O(2) supply to the renal medulla barely exceeds its O(2) requirements. In this study, we examined the impact of the complex structural organization of the rat outer medulla (OM) on O(2) distribution. We extended the region-based mathematical model of the rat OM developed by Layton and Layton (Am J Physiol Renal Physiol 289: F1346-F1366, 2005) to incorporate the transport of RBCs, Hb, and O(2). We considered basal cellular O(2) consumption and O(2) consumption for active transport of NaCl across medullary thick ascending limb epithelia. Our model predicts that the structural organization of the OM results in significant Po(2) gradients in the axial and radial directions. The segregation of descending vasa recta, the main supply of O(2), at the center and immediate periphery of the vascular bundles gives rise to large radial differences in Po(2) between regions, limits O(2) reabsorption from long descending vasa recta, and helps preserve O(2) delivery to the inner medulla. Under baseline conditions, significantly more O(2) is transferred radially between regions by capillary flow, i.e., advection, than by diffusion. In agreement with experimental observations, our results suggest that 79% of the O(2) supplied to the medulla is consumed in the OM and that medullary thick ascending limbs operate on the brink of hypoxia.

A multiunit model of solute and water removal by inner medullary vasa recta

A recent model of volume and solute microcirculatory exchange in the renal medulla based on a single descending vasa rectum (DVR) was extended to account for the varying number of vessels along the corticomedullary axis. The assumption that concentration polarization at the walls of ascending vasa recta (AVR) during volume uptake eliminates transmural oncotic pressure gradients was examined. In this limiting case, small hydrostatic pressure gradients can drive AVR volume uptake if the pressure in the interstitium exceeds that in the AVR lumen. The calculated hydraulic pressure difference across AVR yielding agreement between predicted and measured values of AVR-to-DVR blood flow rate ratios was found to be smaller than the reported maximum pressure difference AVR can sustain. Simulations also confirmed previous conclusions suggesting that the presence of urea transporters in DVR counterbalances that of water channels that would otherwise decrease the efficiency of small solute trapping in the renal medulla.

A mathematical model of O2 transport in the rat outer medulla. II. Impact of outer medullary architecture.

we extended the region-based mathematical model of the urine-concentrating mechanism in the rat outer medulla (OM) developed by Layton and Layton (Am J Physiol Renal Physiol 289: F1346-F1366, 2005) to examine the impact of the complex structural organization of the OM on O(2) transport and distribution. In the present study, we investigated the sensitivity of predicted Po(2) profiles to several parameters that characterize the degree of OM regionalization, boundary conditions, structural dimensions, transmural transport properties, and relative positions and distributions of tubules and vessels. Our results suggest that the fraction of O(2) supplied to descending vasa recta (DVR) that reaches the inner medulla, i.e., a measure of the axial Po(2) gradient in the OM, is insensitive to parameter variations as a result of the sequestration of long DVR in the vascular bundles. In contrast, O(2) distribution among the regions surrounding the vascular core strongly depends on the radial positions of medullary thick ascending limbs (mTALs) relative to the vascular core, the degree of regionalization, and the distribution of short DVR along the corticomedullary axis. Moreover, if it is assumed that the mTAL active Na(+) transport rate decreases when mTAL Po(2) falls below a critical level, O(2) availability to mTALs has a significant impact on the concentrating capability of the model OM. The model also predicts that when the OM undergoes hypertrophy, its concentrating capability increases significantly only when anaerobic metabolism supports a substantial fraction of the mTAL active Na(+) transport and is otherwise critically reduced by low interstitial and mTAL luminal Po(2) in a hypertrophied OM.

Intrinsic nitric oxide and superoxide production regulates descending vasa recta contraction

Descending vasa recta (DVR) are 12- to 15-μm microvessels that supply the renal medulla with blood flow. We examined the ability of intrinsic nitric oxide (NO) and reactive oxygen species (ROS) generation to regulate their vasoactivity. Nitric oxide synthase (NOS) inhibition with N(ω)-nitro-l-arginine methyl ester (l-NAME; 100 μmol/l), or asymmetric N(G),N(G)-dimethyl-l-arginine (ADMA; 100 μmol/l), constricted isolated microperfused DVR by 48.82 ± 4.34 and 27.91 ± 2.91%, respectively. Restoring NO with sodium nitroprusside (SNP; 1 mmol/l) or application of 8-Br-cGMP (100 μmol/l) reversed DVR vasoconstriction by l-NAME. The superoxide dismutase mimetic Tempol (1 mmol/l) and the NAD(P)H inhibitor apocynin (100, 1,000 μmol/l) also blunted ADMA- or l-NAME-induced vasoconstriction, implicating a role for concomitant generation of ROS. A role for ROS generation was also supported by an l-NAME-associated rise in oxidation of dihydroethidium that was prevented by Tempol or apocynin. To test whether H(2)O(2) might play a role, we examined its direct effects. From 1 to 100 μmol/l, H(2)O(2) contracted DVR whereas at 1 mmol/l it was vasodilatory. The H(2)O(2) scavenger polyethylene glycol-catalase reversed H(2)O(2) (10 μmol/l)-induced vasoconstriction; however, it did not affect l-NAME-induced contraction. Finally, the previously known rise in DVR permeability to (22)Na and [(3)H]raffinose that occurs with luminal perfusion was not prevented by NOS blockade. We conclude that intrinsic production of NO and ROS can modulate DVR vasoactivity and that l-NAME-induced vasoconstriction occurs, in part, by modulating superoxide concentration and not through H(2)O(2) generation. Intrinsic NO production does not affect DVR permeability to hydrophilic solutes.

Regulation of pendrin by pH: dependence on glycosylation

Mutations in the anion exchanger pendrin are responsible for Pendred syndrome, an autosomal recessive disease characterized by deafness and goitre. Pendrin is highly expressed in kidney collecting ducts, where it acts as a chloride/bicarbonate exchanger and thereby contributes to the regulation of acid-base homoeostasis and blood pressure. The present study aimed to characterize the intrinsic properties of pendrin. Mouse pendrin was transfected in HEK (human embryonic kidney) 293 and OKP (opossum kidney proximal tubule) cells and its activity was determined by monitoring changes in the intracellular pH induced by variations of transmembrane anion gradients. Combining measurements of pendrin activity with mathematical modelling we found that its affinity for Cl-, HCO3- and OH- varies with intracellular pH, with increased activity at low intracellular pH. Maximal pendrin activity was also stimulated at low extracellular pH, suggesting the presence of both intracellular and extracellular proton regulatory sites. We identified five putative pendrin glycosylation sites, only two of which are used. Mutagenesis-induced disruption of pendrin glycosylation did not alter its cell-surface expression or polarized targeting to the apical membrane and basal activity, but fully abrogated its sensitivity to extracellular pH. The hither to unknown regulation of pendrin by external pH may constitute a key mechanism in controlling ionic exchanges across the collecting duct and inner ear.

Renal Medullary Circulation

The renal medullary microcirculation is a distinctive arrangement of blood vessels that serve multiple functions in the renal medulla. This article begins with a description of the unique anatomy of this vascular bed and the role it plays in transport and countercurrent exchange in the medulla. A segment of the review is then devoted to the important role mathematical modeling has played in the understanding of this vascular beds function. Succeeding sections focus upon the hematocrit in the vasa recta capillaries and methods utilized to assess blood flow in the renal medulla. An extensive portion of the article is then devoted to the regulation of the medullary circulation, from ion channel architecture to neurohormonal signaling. Finally, we discuss the importance of the renal medullary circulation in the regulation of fluid and electrolyte homeostasis and arterial blood pressure regulation.

Membrane current oscillations in descending vasa recta pericytes

We investigated the origin of spontaneous transient inward current (STIC) oscillations in descending vasa recta (DVR) pericytes. In cells clamped at -80 mV, angiotensin II (ANG II; 10 nmol/l) induced oscillations with mean amplitude and frequency of -65.5 pA and 1.2 Hz. Simultaneous recording of cytoplasmic calcium ([Ca(2+)](CYT)) and membrane current oscillations verified their synchrony and the correlation of their amplitudes. Confocal recording in fluo-4-loaded DVR showed that ANG II can induce either stable pericyte [Ca(2+)](CYT) elevation or oscillations, while decreasing adjacent endothelial [Ca(2+)](CYT). Oscillating currents reversed sign at -30.2 mV and were blocked by niflumic acid, implicating charge transfer via Cl(-) ion. Removal of extracellular Ca(2+), blockade of Ca(2+) influx with SKF96365 (30 micromol/l), ryanodine (30 micromol/l), or caffeine (10 mmol/l) inhibited oscillations. In contrast, they were insensitive to removal of extracellular Na(+) and exposure to either nifedipine (1 micromol/l) or 2-aminoethoxydiphenyl borate (10 micromol/l). Ouabain (100 nmol/l) increased basal pericyte [Ca(2+)](CYT) and the frequency of resting STICs but did not affect the larger oscillations that followed ANG II stimulation. We conclude that [Ca(2+)](CYT) oscillations stimulate Cl(-) currents. The former are most likely maintained by repetitive cycles of ryanodine-sensitive SR Ca(2+) release and SKF96365-sensitive store refilling.

Regulation of pendrin by cAMP: possible involvement in β-adrenergic-dependent NaCl retention

The sodium-independent anion exchanger pendrin is expressed in several tissues including the kidney cortical collecting duct (CCD), where it acts as a chloride/bicarbonate exchanger and has been shown to participate in the regulation of acid-base homeostasis and blood pressure. The renal sympathetic nervous system is known to play a key role in the development of salt-induced hypertension. This study aimed to determine whether pendrin may partly mediate the effects of β adrenergic receptors (β-AR) on renal salt handling. We investigated the regulation of pendrin activity by the cAMP/protein kinase A (PKA) signaling pathway, both in vitro in opossum kidney proximal (OKP) cells stably transfected with pendrin cDNA and ex vivo in isolated microperfused CCDs stimulated by isoproterenol, a β-AR agonist. We found that stimulation of the cAMP/PKA pathway in OKP cells increased the amount of pendrin at the cell surface as well as its transport activity. These effects stemmed from increased exocytosis of pendrin and were associated with its phosphorylation. Furthermore, cAMP effects on the membrane expression and activity of pendrin were abolished by mutating the serine 49 located in the intracellular N-terminal domain of pendrin. Finally, we showed that isoproterenol increases pendrin trafficking to the apical membrane as well as the reabsorption of both Cl(-) and Na(+) in microperfused CCDs. All together, our results strongly suggest that pendrin activation by the cAMP/PKA pathway underlies isoproterenol-induced stimulation of NaCl reabsorption in the kidney collecting duct, a mechanism likely involved in the sodium-retaining effect of β-adrenergic agonists.

Chronic potassium depletion increases adrenal progesterone production that is necessary for efficient renal retention of potassium

Modern dietary habits are characterized by high-sodium and low-potassium intakes, each of which was correlated with a higher risk for hypertension. In this study, we examined whether long-term variations in the intake of sodium and potassium induce lasting changes in the plasma concentration of circulating steroids by developing a mathematical model of steroidogenesis in mice. One finding of this model was that mice increase their plasma progesterone levels specifically in response to potassium depletion. This prediction was confirmed by measurements in both male mice and men. Further investigation showed that progesterone regulates renal potassium handling both in males and females under potassium restriction, independent of its role in reproduction. The increase in progesterone production by male mice was time dependent and correlated with decreased urinary potassium content. The progesterone-dependent ability to efficiently retain potassium was because of an RU486 (a progesterone receptor antagonist)-sensitive stimulation of the colonic hydrogen, potassium-ATPase (known as the non-gastric or hydrogen, potassium-ATPase type 2) in the kidney. Thus, in males, a specific progesterone concentration profile induced by chronic potassium restriction regulates potassium balance.