Control of magnesium transport in the thick ascending limb

Transcription

1 Cotrol of magesium trasport i the thick ascedig limb GARY A. QUAMME Departmet of Medicie, Uiversity Hospital, Uiversity of British Columbia, Vacouver, British Columbia V6T 1 W5, Caada QUAMME, GARY A. Cotrol of magesium trasport i the thick ascedig limb. Am. J. Physiol. 256 (Real Fluid Electrolyte Physiol. 25): F197-F210, The mammalia real thick ascedig limb of Hele (TAL) reabsorbs -55% of the filtered magesium; accordigly, it is the major segmet ivolved i cotrol of real Mg balace. This review discusses recet evidece for passive ad active trasport of Mg through the paracellular ad trascellular pathways of the TAL, respectively. The properties of these pathways provide a basis for uderstadig the factors ifluecig magesium reabsorptio ad hormoal cotrols regulatig Mg balace. Normally, Mg absorptio is load depedet, whether delivery is altered by icreasig lumial Mg cocetratio or icreasig the flow rate ito the thick ascedig limb. I cotrast to the lumial cocetratio, elevatio of peritubular (plasma) Mg ad Ca ihibit divalet catio absorptio by mechaisms that are ot etirely clear. Magesium reabsorptio i the TAL is also closely associated with NaCl absorptio so that factors that ifluece NaCl also affect magesium. Magesium deficiecy results i a specific ad apparetly itrisic cellular adaptatio to icrease Mg absorptio i the TAL. Our greatest uderstadig of hormoal cotrols for Mg absorptio have come from recet studies usig a hormoe deprived aimal model. Parathyroid hormoe, calcitoi, glucago, ad atidiuretic hormoe act through a commo secod messeger, adeosie 3,5 -cyclic moophosphate, to limit Mg excretio by ehacig active Mg trasport i the TAL. The itegrated actios of these hormoes ad possibly others provide a sesitive meas of cotrol. Clearly, recet observatios, usig i vivo ad i vitro microperfusio studies, have altered our thikig of TAL fuctio ad idicate that Mg trasport is sesitively ad specifically cotrolled withi this segmet. kidey; loop of Hele; calcium; sodium; active trasport; passive trasport; hypercalcemia; hypermagesemia; furosemide; parathyroid hormoe RECENT STUDIES have clarified our uderstadig of tubular Mg hadlig; particularly importat have bee the observatios cocerig Mg trasport i the thick ascedig limb of the loop of Hele (TAL). The early micropucture studies of Morel, de Rouffigac, Bruette, ad colleagues (8, 16, 44) provided evidece for the importace of the thick ascedig limb i real Mg coservatio. Over 60% of the Mg filtered at the glomerulus is reabsorbed withi the loop of Hele ad it is the reabsorptio withi the loop of Hele ad the epithelial cells of the TAL where the pricipal cotrol of Mg balace appears to be determied (15, 51). Recet elec- trophysiological studies have icreased our uderstadig of thick ascedig limb fuctio with respect to NaCl trasport. The purpose of this review is to discuss some of these recet cotributios ad how they relate to Mg trasport i the TAL ad to idicate some of the uresolved issues that require further ivestigatio. Calcium absorptio will oly be discussed as it relates to Mg trasport withi the TAL. Real hadlig of calcium ad Ca2+ trasport i the TAL have bee reviewed elsewhere (15, 46). Segmetal magesium trasport has bee described with the aid of micropucture techiques i a large /89 $1.50 Copyright the America Physiological Society F197

2 F198 EDITORIAL REVIEW umber of aimal species (16, 35, 37, 54, 70, 71). I all mammals studied to date, Mg is hadled i differet ways alog the ephro segmets. These segmetal characteristics have bee extesively reviewed elsewhere (51). I summary, -80% of the total plasma magesium ( mm) is filtered through the glomerular membrae. Of the ultrafilterable magesium ( mm), 20-25% is reabsorbed by the proximal tubule, icludig the covoluted ad straight portios (Table 1). This is distictive to sodium ad calcium where -70 ad ~60%, respectively, is reabsorbed i the proximal ephro (Table I). Accordigly, the delivery of Mg to the thick ascedig limb of the loop of Hele is relatively much greater tha that of sodium ad calcium. It is ow evidet from micropucture studies that proportioally greater amouts of Mg (50-60%) are reabsorbed i the loop compared with sodium (20-25%) or calcium (30-35%). Because the termial ephro segmets, icludig the distal covoluted tubule ad collectig tubule, reabsorb oly a small portio of the filtered Mg (-5%), the loop of Hele plays a major role i the determiatio of Mg reabsorptio ad it is these segmets where major regulatory factors act to maitai Mg balace (51). Geeral Characteristics of Mg Absorptio i the TAL The evidece for the ivolvemet of the loop of Hele i Mg reabsorptio came from the early i vivo micropucture studies of Morel ad colleagues (44). They collected lumial fluid from the superficial late proximal tubule ad from the early distal tubule of rats ad determied Mg cocetratios with the electro microprobe (44). Magesium cocetratio i the proximal fluid exceeded by as much as 2- to 2.5fold the plasma ultrafilterable cocetratios, whereas the cocetratios i the distal fluid were fractioally lower ( ) tha the ultrafilterable Mg. Compariso of these cocetratios with the respective iuli ratios, a oabsorbable solute used as a marker of water trasport, revealed that some 50-60% of the filtered Mg had bee reabsorbed betwee the late proximal tubule accessible to the surface of the kidey ad the early distal tubule (Table 1). Subsequetly, de Rouffigac et al. (16) i Psammomys obesus ad Bruette et al. (9, 10) i the rat observed a tubular fluid-to-ultrafilterable fluid (TF/UF) cocetratio ratio for Mg at the bed of the loop of Hele i TABLE Segmetal reabsorptio of Mg, Ca, ad Na i ormal aimals excess of that foud at the superficial late proximal tubule (Table 1); thus it became evidet that the ascedig limb was the major site of real Mg coservatio. More recet micropucture studies have supported these earlier fidigs i a umber of aimal species (35, 57). We have examied Mg reabsorptio withi the loop of Hele by i vivo microperfusio techiques (50, 52). The loop also icludes the straight portios of the proximal tubule, the thi ascedig limb, as well as the thick ascedig limb, but it seems likely that the major chages i trasport that were observed occurred withi the TAL (15). The proximal straight tubule absorbs little Mg ad is poorly permeable to Mg (53). The permeability coefficiet of Mg i this segmet is o the order of 1.1 X 10B5 cm/s which is cosiderably lower tha the permeability of either Na+ or Ca2+ (45, 59). This low permeabil- ity to Mg allowed us the opportuity to vary itralumial Mg cocetratio idepedet of the peritubular Mg to assess trasport dowstream i the TAL (52). Accordigly, we could determie Mg trasport over a series of itralumial cocetratios or with various peritubular (plasma) cocetratios. The first experimet was desiged to determie the extet of Mg iflux whe the loops were perfused with Mg2+-free solutios while elevatig peritubular Mg by ifusig MgC12 solutios to the rats. Fluid samples collected at the late distal tubule cotaied oly a small amout of Mg. Although these studies were purposely performed with high flow rates (25 l/mi) to evaluate permeability they suggested that uder these coditios Mg backflux from plasma to lume is small. Levie et al. (39) repeated these studies usig a rage of flow rates ad observed Mg iflux that was depedet o flow rate, i.e., greater iflux at slower flow rates. However, the absolute Mg iflux was much less tha the cocurretly measured sodium or calcium iflux (39). No studies have bee performed to date to assess backflux whe the trasepithelial voltage has bee altered with furosemide or other loop diuretics. These observatios have recetly received support from perfusio studies usig 28Mg to directly determie uidirectioal Mg flux (61). Efflux of Mg (6.95 t 0.7 pmol/mi, = 15 loops) was similar to et Mg absorptio (7.6 t 0.5 pmol/mi). Accordigly, there is ormally egligible backflux of Mg ito the lume, ad Mg absorptio is essetially uidirectioal. I ormal situa- Collectio Site Glomerulus Proximal covoluted tubule Descedig limb of Hele Early distal tubule Late distal tubule Urie 50 Fractio of Filtered Load Remaiig at Collectio Site, % Na+ Ca2+ Miz Data are composite values from Refs. 8, 10, 16, 37, 54, 69, 70, 71 ad represet mea approximatios at the ed of the respective accessible ephro segmets. TF/P ad TF/UF are tubular fluid/plasma or ultrafilterable plasma cocetratio ratios.

3 i i EDITORIAL REVIEW F199.- c E tios the trasepithelial potetial differece is lume positive with respect to the peritubular side, which discourages iflux. These i vivo studies have ot bee performed i the absece of a electrical potetial. The secod series of microerfusio exerimets were performed where lumial MgAcocetratio was elevated (rage mm) at costat flow rates i the presece of ormal plasma Mg ( mm) (52). As metioed above, oe ca do this because of the relatively low trasport rates ad low permeability of Mg i the proximal tubule (50, 52). Net Mg absorptio icreased with r 20 '""T A B Normal Plasma Magesium 9 Hypermagesemia Mg DELIVERY pmoles mi -I, Normal Mg, I I I I I I I I I /- B 36 cl3 8 z 0-l 3.0 0) E = 2.6 m Q 1.8 s I Normal Plasma Magesium 9 Hypermagesemia LUMINAL Mg mm C 0 Normal Plasma Magesium -./. Hypermagesemia I I 1 I 1 I 1.o Na DELIVERY moles. mi -l - --I 5.0 elevatio of lumial Mg so that a Vmax or saturatio of trasport was ot apparet (Fig. 1). Elevatio of lumial Mg by lo-fold above ormal also had o effect o et sodium or calcium trasport withi the TAL (Fig. 1). Next, we asked what effect peritubular Mg has o Mg trasport. Plasma Mg cocetratio was progressively raised while maitaiig the itralumial Mg cocetratio at various fixed levels. Absolute ad fractioal Mg absorptio was markedly ihibited at all lumial cocetratios as the peritubular Mg cocetratio was elevated (Fig. 1). The ihibitio of Mg reabsorptio was directly proportioal to the plasma or peritubular Mg levels (51). Net sodium absorptio was ot affected by peritubular Mg but et calcium trasport was sigificatly reduced (Fig. 1). The practical cosequeces of these observatios were provided by the free-flow micropucture studies i the dog (69). Clearace studies i aimals ad humas have suggested that real Mg hadlig is a Tm-limited process, i.e. possesses a tubular maximum for trasport, which ca be saturated with elevatios of plasma Mg (37,41,58). These studies also implied that humas ad aimals live quite close to this Tm so that ay icrease above ormal plasma Mg levels results i the spillig of Mg ito the urie. However, the micropucture studies i the dog demostrated that real Mg absorptio is ot a saturable process but the summatio of the segmetal reabsorptive rates of the proximal tubule ad the TAL. Mg absorptio is ehaced i the proximal tubule with hypermagesemia due to a icrease i lumial Mg cocetratio (52, 53). Elevatio of plasma Mg does ot ihibit Mg reabsorptio i the proximal tubule but it does i the loop. Accordigly, hypermagesemia results i a icrease i absolute Mg reabsorptio i the proximal tubule ad a decrease i the TAL; the additive effects result i a apparet saturatio or Tm for reabsorptio (51). Mechaisms of NaCl Absorptio i the TAL as it Relates to Mg Trasport How is Mg reabsorbed by the TAL? The greatest cotributios to our uderstadig of tubular Mg hadlig over the last several years comes from our icreased kowledge of the mechaisms of NaCl trasport withi the TAL. These advaces have bee extesively reviewed elsewhere (27,32) but it is pertiet to briefly summarize them here as they pertai to Mg trasport. The TAL is remarkable i that it is characterized by a low permeability to water but a comparatively high permeability to electrolytes. These marked differeces are related to the properties of the paracellular or shut pathway. It has bee suggested that the tight juctios FIG. 1. Effect of lumial Mg vs. peritubular Mg cocetratio o et Mg, Ca, ad Na absorptio i loop of Hele. Loops were perfused at costat flow rates (25 l/mi) ad with solutios cotaiig variable MgC12 cocetratios (O-4 mm) i ormal (ultrafilterable Mg 0.55t 0.03 mm) or hypermagesemic aimals (ultrafilterable Mg, 3.0 t 0.2 mm). Absorptio was calculated by comparig the delivery rates (perfusio rate x electrolyte cocetratio) at late proximal tubule with respective deliveries to accessible early distal covoluted tubule. Mg, Ca, ad Na were quatitated by electro microprobe aalysis ad water absorptio with the use of iuli. Data modified from Quamme ad Dirks (52).

4 F200 EDITORIAL REVIEW of the paracellular pathway allow for electrolyte movemet by sigle-file diffusio but restrict water diffusio (33). Furthermore, the paracellular pathway is catio selective, possibly due to fixed egative charges o the tight juctios (26, 27). Accordigly, small catios may move relatively easily across the epithelium without trasversig the cells. The TAL is composed of two distict portios based o morphological ad fuctioal characteristics: the medullary (MTAL) ad cortical segmets (CTAL). The trasport of NaCl is ormally greater i the medullary segmet tha the cortical portio, but the CTAL maitais greater trasepithelial cocetratio differeces (32). It is thought that a large part of the delivered salt is reabsorbed rapidly i the MTAL where the load is the greatest, but that o large cocetratio differece is created here. Further up the thick ascedig limb, the cortical segmet is able to lower the tubule fluid salt cocetratio perhaps because of its iheretly greater itrisic trasport capacity. Although this otio has recetly bee challeged (27), there is o doubt that each segmet is ormally faced with differig tubular fluid salt cocetratios. Accordigly, there may be sigificat differeces i trasport processes betwee the two segmets although the evidece suggests that the permselectivities of the paracellular pathways are similar. The basis for NaCl absorptio ad the geeratio of a lumial positive electrical potetial is reasoably well uderstood. It is ow kow that Na+-ZCl--K+ trasport is coupled at the lumial membrae of both medullary ad cortical segmets (27). Movemet of. these ios across the lumial membrae occurs due to sodium movig dow its electrochemical gradiet from the tubule fluid ito the cell. The ATP-depedet sodium pump (Na+-K+-ATPase) located at the basolateral or peritubular side of the cell maitais the favorable sodium electrochemical gradiet across the lumial membrae. Sodium exits across the basolateral membrae via the Na+-K+ pump, whereas chloride is thought to move passively from the cell ito the peritubular iterstitium, perhaps coupled i part with potassium (28). Greger (27) has reasoed that the presece of a large potassium coductace across the lumial membrae ad the Clexit across the basolateral membrae may provide the basis for the lume-positive trasepithelial electrical potetial observed i the TAL. I additio to trascellular absorptio, ios may move aroud the cells through the paracellular pathways accordig to the latter s permselective properties. With respect to this model, it is thought that about half of tjhe sodium is trasported through the cell ad the remaider moves passively through the paracellular pathway facilitated by the lume-positive electrical potetial (27, 33). Accordig to this view, the itercellular spaces may be i virtual diffusio equilibrium with the peritubule (32) ad the electromotive force for the trasepithelial voltage is depedet o active cellular Na+-K+ trasport. A positive lumial voltage has bee postulated to be importat i the passive reabsorptio of catios such as sodium, potassium, ad possibly Ca ad Mg (18, 27). This passive movemet has the beeficial effect of reducig the expediture of metabolic eergy for electrolyte reabsorptio (27, 32). But as a cosequece of this teet, factors that ifluece NaCl trasport would be expected to affect the trasepithelial voltage ad thus the passive trasport of Mg ad Ca. Active Versus Passive Mg Trasport CTAL. A umber of direct studies have bee performed usig isolated TAL segmets, which have elighteed our uderstadig of Mg trasport i the loop. Oe importat issue that has bee addressed by a umber of ivestigators is whether Mg trasport is active or passive i ature. The first studies were directed at the cortical portio of the TAL. Shareghi ad Agus (62) perfused CTAL segmets of the rabbit. They varied the trasepithelial voltage from -20 to +30 mv by usig various sodium gradiets across the epithelium, either from lume to bath, or bath to lume i the presece of furosemide to ihibit active Na+-ZCl--K+ trasport. Accordigly, active trasport was ihibited ad the trasepithelial voltage formed was a dilutio potetial depedet o the permselectivity of the paracellular shut pathway for Na+ exceedig Cl-. Mg ios flowed out of the lume (efflux) with a positive lumial potetial ad ito the lume (iflux) with a egative lumial potetial with respect to bath (62). The crossover from efflux to iflux occurred at zero voltage cosistet with passive trasport. However, these studies do ot rule out the presece of active trasport as furosemide may ihibit active Mg movemet as well as secodary active Na+-ZCI--K+ trasport. Nevertheless, these studies would suggest that the electrically leaky TAL is permeable to Mg. Presumably the reaso we did ot see sigificat backflux of Mg i our i vivo perfusio studies (see above) was that the trasepithelial voltage was always egative with respect to the blood. The i situ studies eed to be repeated i the absece of a voltage such as with the iclusio of itralumial furosemide. The early studies of Shareghi ad Agus i the rabbit CTAL have geerally received support from more recet experimets i the rabbit CTAL by DiStefao ad Greger (19, ad persoal commuicatio); ad mouse CTAL by Greger ad de Rouffigac ad colleagues (68, 68a). These ivestigators perfused cortical segmets at low perfusio rates, ad measured et electrolyte movemet, as well as simultaeously determiig the trasepithelial electrical potetial differece (PD) ad resistace values. Basal trasport of Mg was appreciable ad was icreased by -50% with the applicatio of either atidiuretic hormoe (ADH) or glucago. Both hormoes icreased Mg trasport with little, if ay, alteratio i PD, trasepithelial resistace, or NaCl absorptio. Based o the comparatively large chages i et Mg trasport relative to the PD values, these studies suggest that there may be a active compoet of Mg trasport i the CTAL. These iterestig observatios require further support but active ad passive trasport may coexist i the CTAL. Pertiet to the cotroversy surroudig the active

5 EDITORIAL REVIEW F201 ad/or passive ature of Mg trasport are those cocerig the mechaisms of calcium movemet i the CTAL. These cotroversies have bee reviewed extesively elsewhere but it is worth summarizig the saliet poits (18, 46). The early studies of Bourdeau ad Burg i the rabbit CTAL demostrated that Ca flux was highly depedet o the trasepithelial voltage (6, 7). These studies were performed i the presece of furosemide ad used dilutioal salt gradiets to achieve a rage of trasepithelial voltages. Shareghi ad Agus (62) also foud a strog correlatio of et Ca flux with trasepithelial voltage. More recetly, Friedma (25), usig a similar voltage-clamp approach i mouse CTAL reported a sigificat basal rate of et Ca absorptio at zero trasepithelial PD ad symmetrical Ca cocetratios, implyig active Ca trasport. This data is i accordace with the earlier fidigs of Suki et al. (55, 65) i the rabbit CTAL usig isotopic flux ratio aalysis to discer active versus passive trasport. These ivestigators determied that the uidirectioal Ca2+ flux ratios (lumeto-bath/bath-to-lume) exceeded the values predicted by the Ussig flux-ratio equatio, suggestig the presece of active Ca trasport. I these studies furosemide dimiished the trasepithelial voltage but failed to alter Ca efflux. The fidigs of Imai (34) also supported the idea of active Ca trasport but demostrated that furosemide ihibited Ca efflux as well as the voltage. As metioed above, et Ca ad Mg absorptio was observed followig hormoal stimulatio of mouse CTAL without cocommitat chages i trasepithelial PD or resistace, suggestig the presece of active trasport (68, 68a). The geeral cosesus is that active Ca tras- port is preset i rabbit ad mouse CTAL, the two species most ofte used for a source of isolated tubules. The sigificace of the active compoet relative to voltage-depedet trasport remais to be determied. MTAL. Oly two studies have described Mg movemet i the isolated MTAL ad both are prelimiary reports. Shareghi ad Agus (63) observed sigificat et Mg absorptio withi the rabbit MTAL but the ature of this trasport was ot discered. More recet perfusio studies usig isolated mouse MTAL suggests that there is o Mg trasport, either active or passive, i the medullary segmet (68, 68a). Electrolyte trasport was assessed by electro microprobe aalysis of perfusio ad collected fluids. Net Mg absorptio was 0.09 t 0.05 pm01. mi-l. mm+, whereas et NaCl trasport i the medullary segmets was similar to that observed i the CTAL (153 t 17 pmol. mi-. mm-l). Admiistratio of glucago sigificatly ehaced NaCl trasport (226 t 30 pm01. mi- *mm- ) ad PD but had oly a modest effect o Mg absorptio (0.19 t 0.06 pmol. mi. mm-l). These data lead the authors to coclude that little, if ay, Mg was absorbed i the MTAL. These observatios with isolated mouse MTAL segmets were also true for calcium trasport, i.e., virtually o Ca trasport i cotrol (0.06 t 0.07 pmol. mi. mm-l) or followig (0.27 t 0.12) a P.5- to 2-fold icrease i trasepithelial voltage. Accordigly, these prelimiary studies i the mouse suggest that the re are distict differeces i Mg trasport amog regios withi the loop of Hele ad that perhaps the MTAL absorbs little, if ay, Mg (68, 68a). I relatio to calcium movemets, the available evidece suggests that active Ca trasport is abset i the MTAL, i both rabbit ad mouse MTAL (46). Ca efflux is associated with the magitude of trasepithelial PD ad furosemide, which dimiishes voltage results i a decrease i Ca absorptio (25, 46, 64). However, two prelimiary reports have failed to detect ay et Ca (or Mg) movemet i mouse TAL (68, 68a). These discrepacies remai to be resolved. Active ad passive trasport. I summary, there is reasoable evidece for both active ad passive Mg trasport i the CTAL. The evidece for passive trasport is based o the observatio of the depedece of Mg movemet o trasepithelial voltage (28) ad the ihibitio of Mg absorptio by furosemide (49). Support for active Mg trasport has come from recet studies demostratig a stimulatio of Mg movemet by ADH ad glucago i the absece of chages i trasepithelial PD or resistace. It should be metioed that the early studies of Shareghi ad Agus (62) which provided evidece for oly passive movemet, were performed i the absece of hormoes. Accordigly, active trasport may have bee small ad udetectable i this preparatio. No coclusios ca be made at this time cocerig Mg trasport withi the MTAL. Aalogy to Ca trasport would suggest the presece of Mg trasport i this segmet which is voltage depedet, cosoat with the idea of passive, paracellular movemet. However, recet studies from de Rouffigac s ad from Greger s laboratories would suggest that very little Mg trasport occurs i the MTAL. Further studies are ecessary to clarify these issues. Accordigly, Mg2+ may move aroud the cells through the paracellular pathway drive i a passive fashio by the trasepithelial PD ad active sodium trasport. Alteratively, Mg absorptio may be active as well as trascellular i ature. These putative mechaisms have bee schematically illustrated i Fig. 2. The quatitative aspects of these trasport pathways are far from clear. Trascellular absorptio would ivolve at least oe higheergy barrier, the basolateral membrae. Itracellular Mg2+ cocetratio has bee reported to be i the rage of mm (30, 66). Accordigly, Mg2+ may eter the cells dow a electrical gradiet ad be actively extruded across the basolateral membrae. The meas by which Mg2+ actively moves across the basolateral membrae is ukow. Evidece take from studies with oepithelial cells idicates that Na+-Mg2+ exchage may occur; Na+ movig back ito the cell may be coupled with Mg2+ trasport from the cell ito the iterstitium (4, 24, 29). Alteratively, a specific eergy-depedet Mg pump may be preset aalogous to that reported for Ca (52) (Fig. 2). Accordig to this hypothetical model, both passive paracellular movemet ad active trascellular trasport would ultimately be depedet o active NaCl absorptio. The presece of both putative passive ad active meas of Mg trasport explais may of the regulatory cotrols ad iflueces detailed below.

6 F202 EDITORIAL REVIEW Na+ Lume 1 Cell 1 Blood PD+l OmV -7OmV 70mV OmV Mg mM PD+l MCI : OmV l *. Mg2+ -1 mm 0.5mM Mg 2+ -7OmV -7OmV OmV I FIG. 2. Schematic model of Mg absorptio i thick ascedig limb of Hele s Loop. Coductive pathways are deoted by dashed arrows ad carrier-mediated trasport by solid arrows. Active trasport processes are idicated by - symbol. Modified from Greger (27) ad Hebert ad Adreoli (32). Adaptatio to Mg Deficiecy The kidey has a remarkable capacity to coserve Mg ad limit the amout of Mg excreted i the urie durig Mg-deficiet states (11, 50). This respose is rapid (withi hours) ad appears to be characteristic of the loop of Hele ad the cells of the thick ascedig limb (11, 50, 56). The questio whether this is simply load depedece of Mg trasport with delivery, i.e., fall i lumial Mg cocetratio, or represets a importat adjustmet i trasport rate remais uclear. The fall i uriary Mg excretio rate precedes the fall i plasma Mg cocetratio (upublished observatios). As the GFR remais uchaged, it suggests that Mg coserva- tio is ot etirely depedet o the filtered load o plasma cocetratio. Recetly, we have performed studies o a isolated epithelial cell lie to address the issue of whether cells may itrisically alter their trasport rates i respose to ambiet Mg cocetratio. The cell lie used was the opossum kidey cell (OK cell lie), which is most likely derived from the proximal tubule. Nevertheless, it was used because it expresses regulatory cotrols for various trasport processes (40). Cells maitaied o a low-mg (CO.01 mm) media trasported 1.4- to U-fold more Mg tha cells maitaied o ormal-mg (0.75 mm) media (upublished observatios). These studies idicated that epithelial cells may itrisically respod to low evirometal Mg levels by up-regulatio of Mg trasport. The ability to itrisically adapt to low Mg may also be a property of TAL cells. Load Depedece of Mg Absorptio i the TAL It is well established that there is a depedece of NaCl trasport rate with the load of salt delivered to the loop of Hele (27, 72). This is also true for Mg absorptio i the loop (Fig. 1). The balace betwee absolute absorptio ad load is maitaied whether the delivery is altered either by lumial Mg cocetratio (50, 52) or by variatio i the flow rate to the loop (61). Evidece substatiatig this coclusio is derived from i vivo microperfusio studies (Fig. 1 ad Fig. 3). Loops were perfused from a proximal tubule site ad lumial fluid sampled from the earliest accessible distal tubule. Perfusio rates were varied from 15 to 40 l/mi to determie the effect of flow rate vs. lumial Mg cocetratio. Similar uidirectioal Mg flux rates were observed for equal delivered loads whether it was by alterig flow rate at costat lumial cocetratios or by chagig lumial cocetratios with costat perfusio rates. Fractioal reabsorptio amouted to -80% with ormal delivery rates. Flow rates much greater tha 30 l/mi, i.e., above physiological, resulted i dimiished absolute Mg trasport cocurret, ad possibly associated, with the fall i NaCl trasport (Fig. 3). The basis for the balace betwee trasport ad delivery is presumably similar to that used to explai flow-depedet NaCl trasport (27,72). By aalogy to sodium, we might expect that icreases i ascedig limb flow rate may cause Mg absorptio to icrease because icreases i flow rate cause the Mg cocetratio i tubule fluid to icrease alog the legth of the TAL (72). It is the dissipatio of the cocetratio gradiet ad the geeratio of a dilute fluid with respect to Mg that determies et trasport. It is likely that both active ad passive terms are affected by cocetratio chages. Accordigly, the balace betwee flow rate ad absorptio i the TAL is idepedet of the ature of passive or active Mg trasport. However, the set poit for the load depedece is most likely determied by factors alterig trasepithelial voltage (NaCl) ad/or active Mg trasport. I the absece of extrisic factors, either peritubular iflueces or hormoal cotrols, the loop has a itrisic ability to limit the excretio of Mg. Accordigly, a modest icrease i delivery, for example followig the ihibitio of proximal tubular reabsorptio by extracellular volume expasio, results i a greater absolute absorptio i the loop of Hele, which teds to mitigate the chages i uriary excretio. Large icreases i volume flow that exceed the bufferig ability of the loop must ultimately lead to icreases i uriary excretio. Associatio of Mg Trasport with NaCl Absorptio i the TAL Active Na trasport. As previously metioed, there is reasoable evidece to idicate that Mg trasport i the

7 EDITORIAL REVIEW F203 C FLOW RATE l. mi -1 FLOW RATE l. mi -1 t I I t I I t tive trascellular movemet of Mg as schematically depicted i Fig. 2, would also be expected to be iflueced by chages i active NaCl trasport. Alteratio of lumial or basolateral trasmembrae electrochemical potetial for sodium may affect Mg uptake ad extrusio if these membrae evets are drive by passive or secodary active meas. Accordigly, paracellular or trascellular Mg absorptio may be closely associated with active sodium trasport i the TAL. Loop diuretics. Diuretics, such as furosemide ad bumetaide, dimiish salt absorptio i the TAL by virtue of their actio o electroeutral Na+-2Cl--K+ cotrasport across the lumial membrae (27). Ihibitio of lumial Cl- etry leads to dimiished cellular Cl- activity ad dimiished basolateral coductive Cl- efflux which results i a decrease i lume-positive voltage (27, 32). It has bee postulated that the lume-positive voltage provides the drivig force through the paracellular route for 40~50% of the total et Na+ absorptio. Mg, a divalet catio, may be iflueced to a greater degree tha the moovalet catios, such as Na+ ad K+. This otio is supported by the fidigs preseted i Fig. 4, which illustrates that et Mg trasport is ihibited to a greater degree tha Na at ay give lumial furosemide cocetratio. I additio to chagig the trasepithelial voltage, furosemide may act through other mechaisms o Mg trasport, possibly trascellular. The observatio of fractioally greater ihibitio of TAL Mg absorptio compared with sodium is relevat to the maagemet of patiets receivig loop diuretics, because hypomagesemia ad hypocalcemia are possible complicatios of diuretic therapy. A iterestig cosequece of furosemide s actio o lumial Na+-2Cl--K+ cotrasport is that trascellular movemet of Mg2+ may actually be accelerated as the passive voltage-drive compoet is dimiished. Ihibitio of apical Na+-2Cl--K+ cotrasport may hyperpolarize the basolateral membrae thereby facilitatig Mg etry ad ehacig active basolateral extrusio. Some evidece has bee give i support of this idea with respect to Ca trasport i the CTAL (25, 64). 1 ; I I I I FLOW RATE l mi -1 I i FIG. 3. Associatio of et Mg trasport with flow rate ito thi ascedig limb of Hele (TAL). Superficial rat TAL were perfused from proximal tubule at various flow rates but with same electrolyte cocetratios. Collectios were performed from earliest accessible distal tubule. Details of experimetal methodology are give i Ref. 52. TAL is, i part, passive i ature ad depedet o the trasepithelial voltage. Accordigly, ay ifluece that affects the trasepithelial electrical potetial, either through chages i active NaCl trasport or by alteratio i the permeability of the paracellular pathway, will affect et Mg absorptio. Furthermore, the putative ac- Cotrol 3x1 O-6M 3x1 O-5M 15~1O-~M FIG. 4. Effect of itralumial furosemide o fractioal electrolyte reabsorptio withi perfused loop of Hele. Superficial thi ascedig limbs (TAL) were perfused with ormal Riger solutio at 25 l/mi. ** P < 0.01 compared with Na. * P < 0.01 compared with Ca. [From Quamme (49).]

8 F204 EDITORIAL REVIEW Hypermagesemia ad Hypercalcemia Elevatio of either plasma Mg or plasma Ca ihibits et Mg trasport i the TAL (9, 37, 38, 48, 52, 69). This iteractio lead early ivestigators to suggest that Ca2+ ad Mg2+ may share the same trasport system (67). This is clearly ot the case, as itralumial Mg or Ca does ot ihibit the respective trasport rates, i.e., elevatio of itralumial Mg did ot ihibit Ca absorptio (52) or did elevatio of itralumial Ca ihibit Mg trasport (48) (Fig. 1). However, elevatio of plasma ad peritubular cocetratio of either Ca or Mg ihibited both et Mg ad Ca absorptio (Fig. 1). Recet i vivo microperfusio studies usig Mg to determie uidirectioal flux rates have idicated that this dimiished et trasport is due to the ihibitio of uidirectioal Mg efflux rather tha a icrease i the iflux rates (61). Shareghi ad Agus (62) determied the effects of lumial vs. bath Mg ad Ca i the isolated perfused rabbit CTAL. Elevatio of lumial Mg by twofold (0.70 to 1.40 mm) app lroximately doubled et Mg absorptio but had o effect o calcium trasport. Icreasig bath Mg cocetratio by similar amouts completely ihibited Mg trasport with o alteratio i Ca absorptio. Similar observatios were reported whe lumial ad bath Ca cocetratios were chaged idepedetly. Elevatio of lumial Ca (1.25 to 2.50 mm) resulted i a twofold icrease i Ca absorptio, whereas elevatio of bath Ca levels ihibited et Ca absorptio by -80%. No effects were observed i Mg trasport with either lumial or bath Ca. Elevatio of bath, but ot lumial, Mg ad Ca resulted i a modest declie i trasepithelial PD (lo- 15%). These data lead the authors to coclude that bath Mg ad Ca dimiish the respective divalet catio absorptive rates, whereas there is o i hibitio by elevatio of the lumial cocetratio; ideed trasport was highly depedet o lumial cocetratio. These observatios were cosoat with the idea of separate Mg ad Ca pathways i the TAL. However, they are at odds with the observatios of micropucture studies i the itact TAL (9, 37, 49, 52, 69) idicatig that plasma Mg ad Ca alter both Mg ad Ca trasport. The site of iteractio of plasma Mg ad Ca o Mg ad Ca absorptio, followig hypermagesemia ad hypercalcemia, may reside i the mtal (63). A alterative explaatio has bee advaced to explai the ihibitio of Mg ad Ca trasport i the TAL by hypermagesemia ad hypercalcemia (27). This explaatio is based o the kow effects of Ca o the tight juctios of epithelia such as the gallbladder (17, 73). This postulate ivolves the titratio of fixed egative charges of the tight juctio of the paracellular pathway by Ca2+ ad Mg2+, leadig to a icrease i epithelial resistace as evideced by a observed decrease i PNa/ PC1 (differetial permeability of sodium to chloride) ratio with icreased Ca2+ or Mg2+ cocetratios (73). A icrease i resistace to catios, specifically divalet catios, would decrease voltage-depedet absorptio through the paracellular pathway. Data from recet microperfusio studies, with isolated rabbit CTAL segmets, have supported this otio (27, ad persoal commuicatio). Elevatio of either lumial or bath Ca ad Mg (from 2.5 to 10 mm) icreased trasepithelial resistace by 50% (from 36.0 t 2.0 to 54.0 t 5.6 Q.cm2). Further studies, based o the effects of Mg2+ ad Ca2+ o NaCl dilutio potetials (i the presece of furosemide), idicated that this resistace chage was associ- ated with a decrease i P&&I (from -3.0 to 2.0). The authors speculate that these chages would oly margially alter NaCl trasport but may markedly alter divalet catio absorptio; the latter were ot directly determied i these studies (19). If these coclusios are valid, it would explai the observatios withi the itact kidey that hypermagesemia ad hypercalciuria result i large icreases i uriary Mg ad Ca excretio with miimal alteratios i NaCl excretio. However, they do ot explai our earlier data (52), which idicated that the iteractio was asymmetrical, i.e., oly peritubular cocetratio was effective i dimiishig trasport (Fig. 1). The discrepacy betwee the i vitro microperfusios ad the i vivo experimets may be due to the differeces i the cocetratios of divalet catios used i the respective studies. Figure 1 illustrates the asymmetrical actios of lumial Mg vs. peritubular Mg cocetratio o et Mg, Ca, ad Na absorptio withi the perfused superficial rat TAL. Lumial Mg cocetratios i excess of fivefold those ormally observed i the itact loop did ot ihibit et Mg, Ca, or Na absorptio. Net Mg absorptio was highly depedet o lumial cocetratio so that fractioal reabsorptio (75-80%) remaied uchaged. However, elevatio of peritubular Mg by about threefold, resulted i markedly dimiished fractioal Mg ad Ca absorptio to 32 $- 4 ad 56 t 3%, respectively. Studies were performed to evaluate greater lumial Mg cocetratios; this was ot possible from the peritubular side as plasma Mg levels much above 4-5 mm were lethal to the aimal. Elevatio of lumial Mg (from 0.5 to 10 mm) resulted i dimiished fractioal Mg (22%), Ca2 (54%), ad Na+ (75%) absorptio (Fig. 5). Iterestigly, the iflectio poit, i.e., the lumial Mg cocetratio that resulted i half-maximal ihibitio was similar for all three catios, suggestig a commo effect o the three catios. This may imply a alteratio i trasepithelial resistace ad decreased movemet through the paracellular pathway. Although a defiitive explaatio is ot possible, lumial cocetratios ecessary to alter trasport grossly exceed those ormally observed i hyper- magesemia. Coversely, elevatio of peritubular Mg ihibits Mg ad Ca i the physiological cocetratio rage for these catios. I summary, it is clear that hypermagesemia or hypercalcemia result i elevated uriary excretio of both Mg ad Ca. The mechaisms are uclear but the preset evidece suggests that the iteractios are greater from the peritubular tha the lumial side. This asymmetrical actio of lumial vs. peritubular Mg may ivolve ad/or paracellular aspects of Mg movemet. Hormoal Cotrol of TAL Mg Absorptio active Magesium absorptio. It has bee kow for some time that parathyroid hormoe (PTH) ehaces tubular

9 l l l l EDITORIAL REVIEW F B 90 R z 60 $ 50 r \ 80 E 100 i= k 0 q, q.--_--mm-- e 0 q Ido q o 0 II q 0 III I I I I I I I I I I I I N :molmg a Ma DELIVERY Dmoles mi -l i?, w \\ \ -2. I = + ---_ 10 m -m--, _ ,a mm m.. 0 I I 1 I I No:mal,Mg Mg DELIVERY pmoles. mi LUMINAL Mg mm LUMINAL Mg mm 0 q 0 q D I 10 I I I I I I I I l I I l I I I I I I I I I I I I 0 II Mg DELIVERY pmoles mi -l Normal Mg L J LUMINAL Mg mm l \ -I :\., - l, \ I \ \ 8. \ \ \. \ -8 =\ *, t l l _ -m N ormal I Mg Mg DELIVERY pmoles. mi m I. 8 8 l 60 I I I I I I I I I I I I I I I I I I I I I I I Mg DELIVERY pmoles. mi -1 Mg DELIVERY pmoles. mi -l No,rmal,Mg Normal Mg I I r LUMINAL Mg mm LUMINAL Mg mm FIG. 5. Effect of lumial Mg o fractioal Mg, Ca, ad Na absorptio rates withi thi ascedig limb (TAL). Loops were perfused i situ with solutios cotaiig MgC12 at costat perfusio rates. Rage of ormal lumial Mg cocetratios is less tha 2.0 as idicated o abscissa. Cocetratios markedly i excess of ormal are required to alter trasport rates (52).

10 F206 EDITORIAL REVIEW reabsorptio of Mg ad dimiishes uriary Mg excretio Cl (41, 51, 67). May of the early micropucture studies were performed o thyroparathyroidectomized aimals ad the data compared with subsequet collectios fol lowig hormoe replacemet. The hormoal effects were 0.2 margial or ofte ot detected (36, 52), ad may required special circumstaces to demostrate segmetal 0.1 actio (41, 49, 50, 69). However, the golde hamster has z o ;I c E prove to be a useful model to study PTH actio (31). $ Thyroparathyroidectomized hamsters excrete -14% of t the filtered Mg i the urie. Followig PTH admiistra- *i tio, fractioal Mg excretio falls to 2-3% due to stim-. * k 0.3- ulatio of Mg reabsorptio withi the TAL; as reflected % by the decrease i distal TF/UF ratios from j 0.2- to 0.33 t PTH has also bee show to icrease Mg E O.l- Na WI Ca 1 CE -+I c + absorptio beyod the TAL, i the distal covoluted E CE i_ tubule (2, 3, 21, 31, 50, 51, 69). The exact ature of this - z OS hormoal actio remais to be clarified. The specificity I of actio is also debatable as PTH also stimulates Ca reabsorptio i these ephro segmets. Nevertheless, curret evidece idicates that PTH, actig i cocert with other hormoal factors metioed below, is suffi- E! 0.2 z ciet for adequate cotrol of real Mg balace. : 0.1 A large umber of hormoes act withi the TAL; may - k CE of which have bee demostrated to alter Mg absorptio 0 (1, 21, 22, 47). Recet studies have added greatly to our F A l-m uderstadig of hormoal cotrols for Mg hadlig, I I l-7 J r withi the TAL (15). Morel et al. (43) showed that PTH, calcitoi, ad glucago stimulate the geeratio of CAMP i the CTAL, whereas ADH, calcitoi, ad glu- cago release the same secod messeger i the MTAL. Moreover, these hormoes appear to act o the same adeylyl cyclase pool withi the target cell. Accordigly, each oe of these hormoes should lead to similar fuctioal resposes predicted o receptor distributio withi each segmet (42). De Rouffigac ad colleagues developed a ovel approach to test this theory (12, 14). They used Brattleboro rats with hereditary diabetes isipidus, which lack edogeous ADH, ad they ifused either glucose or somatostati to ihibit glucago secretio. Furthermore, they thyroparathyroidectomized the aimals to elimiate circulatig PTH ad calcitoi. Thus a hormoe-deprived aimal model was created to serve as a basis for evaluatig the respective actios of each hormoe. Micropucture studies were the performed to determie the sigle ad combied effects of hormoe admiistratio; importatly these studies were all performed with physiological hormoe cocetratios. Figure 6 summarizes the effect of each of the hormoes o the compositio of tubular fluid collected from the early distal tubule (15). As there was o, or little, effect of these hormoes o proximal tubular fuctio, the chages observed i the distal fluid reflect alteratios withi the TAL. Moreover, the chages i fractioal uriary excretio were associated with those observed withi the TAL for each hormoe. All four hormoes, PTH, glucago, calcitoi, ad the ADH aalogue, 1-desamio&D-argiie vasopressi, reduced the cocetratio of Mg ad Ca i distally collected tubular fluid as a cosequece of ehaced reabsorptio 0 ILL CE t CE * f : 3 CE ddavp Calcito Glucago E ^ FIG. 6. Effects of 1-desamio-8-D-argiie vasopressi (ddavp), calcitoi, glucago, ad parathyroid hormoe (PTH) o Cl, Na, Mg, Ca, ad K tubular-fluid-to-plasma ultrafilterate cocetratio ratios (TF/UF) i early distal tubules. Experimets were performed i hormoe-deprived rats (Brattleboro diabetes isipidus rats that were acutely thyroparathyroidectomized ad ifused with somatostati to ihibit glucago secretio) receivig oe of the lackig peptides. Admiistratio rates of peptides were (per 100 g body wt): ddavp, 20 pg/mi; huma calcitoi, 1 mu/mi; glucago, 5 g/mi; PTH, 5 mu/mi. Abbreviatios ad symbols are C, cotrol; E, experimetal, * P < 0.05, ** P < Each colum represets mea value obtaied from 5 to 6 differet aimals. [From De Rouffigac et al. (15).] i the loop of Hele (1, 22, 23). Calcitoi, ADH, ad glucago, but ot PTH stimulated NaCl trasport i the TAL. These authors also showed that may of the actios of these hormoes were additive (20). Thus oe hormoe may stimulate Mg trasport i the presece of the others. For example, the ifusio of supramaximal doses of glucago ad ADH to hormoe-deprived aimals resulted i greater reabsorptio compared with aimals receivig either hormoe aloe (Fig. 7). Accordigly, the cocerted actio of two or more hormoes may be importat i cotrollig TAL fuctios. Further studies of this ature are warrated to firmly establish the importace of multiple hormoal cotrol. Oce it became evidet that idividual hormoes acted o the same adeylyl cyclase pool ad that oe could discer idividual hormoe actios, it was possible to i

11 EDITORIAL REVIEW F T time (mi) FIG. 7. Additive effects of glucago ad atidiuretic hormoe (ADH) o real Mg reabsorptio. Fractioal uriary Mg excretio is expressed over time i hormoe-deprived rats give ADH aloe, 40 pg/ mi (0) or ADH plus glucago, 1 g/mi, (a) or ADH plus glucago 10 g/mi (0). * Sigificace from rats give ADH aloe ad ** rats give ADH plus glucago, 1 g/mi ( P c 0.05). [From Elalouf et al. (20).] use this iformatio together with kowledge of receptor distributio to speculate o segmetal itegratio of hormoe actio. Parathyroid hormoe stimulates adeosie 3,5 -cyclic moophosphate (CAMP) productio i the rat CTAL but ot i the MTAL. PTH does ot alter NaCl absorptio i the hormoe-derived rat, supportig the otio that PTH stimulates Mg trasport i the CTAL without alteratio of NaCl absorptio. Calcitoi, glucago, ad ADH, o the other had, stimulate CAMP release i CTAL ad MTAL ad ehaces NaCl absorptio as well as Mg. By iferece, calcitoi, glucago, ad ADH may act o CTAL to icrease active Mg trasport ad o MTAL to ehace NaCl uptake. Although all four hormoes chage Mg trasport rate it is apparet that segmetal heterogeeity is preset. Nevertheless, i situ studies such as these are importat to provide a itegrated picture of TAL fuctio (5,X,47). Oly a few i vitro studies have bee performed to isolate the site ad mechaism of actio. These studies have ecessarily bee performed o species other tha the rat because of the ease of obtaiig isolated tubule segmets. Shareghi ad Agus (62) observed a 1.5 to 2.0- fold icrease i Mg ad Ca absorptio i rabbit CTAL without a chage i the trasepithelial PD or alteratio of NaCl trasport followig the additio of PTH to the bathig solutio. De Rouffigac et al. (68a) reported that ADH icreased et Mg ad Ca absorptio by 9 ad 38%, respectively, i mouse CTAL i the absece of alteratios i trasepithelial voltage or resistace measuremets. I parallel experimets, glucago icreased Mg ad Ca flux by 1.5- to Z.&fold ad NaCl by lo-15% with o effect o cocurretly measured trasepithelial PD or resistace parameters (68). There was o observable stimulatio of Mg trasport i the MTAL i these latter studies (68); absorptio remaied essetially zero despite the presece of appreciable NaCl flux. These data are etirely harmoious with the fidigs obtaied i the hormoe-deprived rat model. No studies have bee performed to determie the additive actios of these hormoes o Mg trasport i isolated CTAL segmets. Calcium absorptio. It may be germaie to briefly * review some of the hormoal effects o calcium absorptio i isolated CTAL ad MTAL segmets as more studies have bee directed at Ca hadlig tha Mg trasport. A umber of ivestigators have show that PTH stimulates et Ca absorptio i the isolated CTAL (7, 25, 64, 65). However, the mechaisms of actio are cotroversial. Bourdeau ad Burg (7) reported that PTH ad CAMP may ehace Ca trasport i rabbit CTAL by facilitatig passive, voltage-drive, Ca diffusio presumably by alterig the permeability properties of the paracellular pathway. Suki et al. (64, 65), o the other had, provided evidece for hormoal stimulatio of trascellular Ca flux. The latter studies were supported by the fidigs of Imai et al. (34). The basis for these diverget views has bee discussed extesively elsewhere (7, 25, 46). More recetly, Friedma (25) has provided evidece i the mouse CTAL that Ca absorptio is active ad stimulated by PTH ad CAMP. These studies were performed i the presece of furosemide or bumetamide to elimiate active NaCl trasport. Ca absorptio was evidet i the absece of a trasepithelial voltage ad was stimulated by additio of PTH to the bathig solutio (25). O balace, these studies all idicate that PTH ehaces Ca trasport i the CTAL with strog evidece for stimulatio of active trasport. Other studies have demostrated that atidiuretic hormoe (68a) ad glucago (67) but ot calcitoi (46) icrease Ca trasport i isolated CTAL, ledig credace to the otio that CAMP-mediated actio is importat i Ca coservatio. The MTAL of the rabbit ad mouse lack PTH-sesitive adeylyl cyclase; accordigly, there is o stimulatio of Ca trasport i this segmet. However, calcitoi (46) ad CAMP (25) may stimulate Ca absorptio i rabbit ad mouse MTAL, respectively, apparetly by icreasig the voltage-depedet passive movemet. Coversely, other studies have idicated that glucago ad ADH ehace NaCl trasport ad the PD but have little effect o basal Ca trasport (68,68a). The discrepacies amog these studies remai uexplaied. CeZluZar actios of CAMP. It is ow clear that hormoes actig through the secod messeger, CAMP, appear to play a importat role i orchestratig real Mg balace through cocerted actios o the TAL (15). Recet elec- trophysiological studies idicate that CAMP does ot affect the passive permeability properties of the TAL; accordigly, the icrease i trasepithial coductace is apparetly due to a icrease i trascellular NaCl movemet rather tha alteratios i the shut pathway (32, 60). The meas by which itracellular CAMP stimulates Mg trasport is ukow. It is thought that CAMP may icrease basolateral Cl- coductace ad secodarily ehace potassium efflux across the apical mem- brae, which would cotribute to hormoe-depedet icrease i trascellular NaCl movemet (27, 28). The associated icrease i trasepithelial voltage would icrease paracellular Mg movemet i those segmets that demostrate passive trasport. A icrease i trascellular Mg trasport i the absece of trasepithelial elec- trochemical gradiets is more difficult to explai. A icrease i Mg2+ ad Ca2+ without associated icreases