Department of Pharmacology, National Cardiovascular Center Research Institute, Osaka 565 and Department of Cardiology, Jichi MedicalSchool, Tochigi 329-04, Japan
The existence of a steep osmotic gradient in the renalmedullary interstitium is the most critical in the formation ofconcentrated urine [1]. The architectural organization of therenal tubules and blood vessels in the medulla constitutescounterfiow systems which are essential for both generating andmaintaining high osmotic pressure of the renal medulla [2—4].While it has been generally accepted that active NaCl transportin the thick ascending limb of Henle's loop plays the mostfundamental role in the operation of the countercurrent multi-plication system in the renal medulla, it is still a matter ofconsiderable dispute whether the thin ascending limb (tAL) alsohas an active salt transport system to provide a "single effect"necessary for the operation of the countercurrent multiplicationsystem.
However, the single solute model utilizing active NaCl trans-port along the ascending limbs of Henle's loop for a "singleeffect" [5] was not sufficient to explain the significant contribu-tion of urea to the formation of concentrated urine [6—15]. Inaddition, this model did not acknowledge some obvious mor-phological (and possibly functional) differences between thethick and thin segments of the ascending limbs. The modelsproposed by Stephenson [16] and by Kokko and Rector [17]incorporated these two important features. According to thesemodels, unique membrane characteristics are required for thethin ascending limb, that is, the segment should be impermeableto water, highly permeable to NaCl and less permeable to urea.These transport characteristics allow outward diffusion of NaC1in excess of inward diffusion of urea, thereby conforming to the"single effect" generating the relative hypotonicity of theluminal fluid without active solute transport.
Although these essential features of the model have beenverified by in vitro microperfusion studies [18—20], many com-puter simulation studies [21—23] failed to generate an osmoticgradient in the inner medulla in the absence of active solutetransport in the tAL. The most difficult problem in the model ofKokko and Rector [17] is that the multiplication system doesnot work if urea concentration of the tubular fluid at the hairpinturn is increased by addition of urea along the descending limb.However, this does not necessarily negate the validity of thesolute mixing models, but rather suggests that some fundamen-tal information is lacking for the computer analysis. The passivemodel is very attractive because it utilizes energy efficiently and
explains the role of urea. The purpose of this communication isto review the transport properties of the thin segments ofHenle's loop in an attempt to seek the possibility of generatinga new idea for the countercurrent multiplication system withoutactive solute transport.
Anatomical aspects
Elegant morphologic studies reported in the last decade[24—36] have disclosed that there are considerable inter- andintranephron heterogeneity and species differences in the mor-phology and architecture of the thin loop segments and bloodvessels in the renal medulla. Since such anatomical featureshave already been reviewed in detail [2—4] and are also dis-cussed in this symposium [37], we will briefly summarize onlythose features which are relevant to the later discussion on thefunction of the thin segments of Henle's loop (Fig. 1).
Epithelia of thin segments of Henle's loopGenerally, we can divide nephrons into two groups: those
with short and those with long loops (Fig. 1). The thin limb ofthe short—loop nephron is confined to the descending limb,whereas thin limb segments of the long—loop nephron consist ofboth descending and thin ascending limbs. Marked inter- andintranephron heterogeneities are noted in the descending limbof Henle in some rodents, including mice [25], rats [24, 26, 32],Psammomys obesus [28, 29], Octodon degus [27], Perognathuspenicillatus [35], and hamsters [36]. Descending limbs ofshort—loop nephron (short descending limb, SDL) have simple,flat and noninterdigitating epithelia that contain sparse cellorganelles. They are called type I epithelia [25, 28].
The morphology of the tight junctions in the SDL is charac-terized by a deep junctional complex which contains severalramified junctional strands [32, 33, 36]. These are characteristicof the 'tight" tight—junction.
On the other hand, the structure of the descending limb of thelong—loop nephron (long descending limb, LDL) is more com-plicated in that there is intranephron heterogeneity in themorphology of epithelia. The upper portion of this segment(LDL) consists of more differentiated epithelia with compli-cated basolateral interdigitations and apical microvilli calledtype II epithelia [25, 29]. In hamster kidney there is even a moreelaborate epithelium called type ha [361. The tight junctions ofepithelia in the LDLu are characterized by a shallow junctionalcomplex containing a simple junctional strand, characteristic ofthe "leaky" tight—junction. The tubular diameter and epithelialheight are much greater in the LDLu than in the SDL.
565
566 Imai et a!
Fig. 1. Schematic illustration of epithelial morphology of thin loopsegments and architectural organization of the renal medulla. (I),short—loop descending limb (SDL); (II), upper portion of long—loopdescending limb (LDLu); (III), lower portion of long—loop descendinglimb (LDLL); (IV), thin ascending limb. Epithelial morphology labeled(II') represents the rabbit LDL. VB denotes vascular bundle.
The lower part of long descending limbs (LDLL) is lined byflat, noninterdigitating, simple epithelia, which are very similarto SDL epithelia and are called type III epithelia [25, 29]. Thetransition from the epithelium of LDL to that of LDLL occursat various levels from the outer to inner medulla [2, 3, 30].
In marked contrast to the descending limbs of these species,there is much less heterogeneity of the descending limbs of therabbit nephron [31]. Although the epithelia of the LDLu ofrabbit kidney are more differentiated than those of the SDL orLDLL, they scarcely interdigitate and the tight junctions aredeep. It is clear that there are interspecies as well as inter-nephron differences in the morphology of the descending limb.
On the other hand, the structure of the thin ascending limb(tAL) is very similar among various species including rats [24,26, 32], mice [25], Psammomys obesus [29], Octodon degus[27], rabbits [31], golden hamsters [36], and cats [3] (Fig. 1). Theepithelia of the tAL are characterized by very flat cells withcomplicated interdigitations. They are called type IV epithelia[25, 29]. Tight junctions are very shallow, characteristic of a"loose" tight—junction.
Tubulo-vascular relationship in the renal medullaThe nephron segments and blood vessels in the renal medulla
exhibit a unique architectural organization, which is also con-sidered to be important for the urinary concentrating mecha-nism [2—4, 38—43]. In principle, a simple countercurrent systemconsists of two limbs side by side with each other, with the endof these limbs connected to form a hairpin turn. However, evenin the avian kidney, which has the most primitive form of thecountercurrent system, we do not encounter such a simole
countercurrent pair [38]. The descending limbs of avian kidney,distributed mainly in the center of the medullary cone, areclearly separated by interposed collecting ducts from the thickascending limbs which are mainly positioned in the periphery.A similar separation of thin descending limbs of short—loopnephron from thick ascending limbs is also encountered in thekidney of many mammalian species which have the so—calledcomplex type of vascular bundle [2—4], where the SDL isintegrated in the vascular bundle. These species include the rat[39], mouse [40], Meriones shawii [41] and Psammomys obesus[42, 43]. It should be noted that these species have a highurinary concentrating capacity.
Another unique feature of the tubulo-vascular organization inthe renal medulla is the fact that in all species the blood flow ofinner stripe of outer medulla and that of inner medulla arealmost completely separated [2—4]. This was carefully empha-sized by Jamison and Kriz [3], who described that "Bloodflowing to the inner medulla has not previously been exposed totubules of the inner or outer stripe" and that "Blood which hasperfused tubules of the inner medulla does not then perfusetubules of the inner stripe." These descriptions suggest that thelong—loop nephron is functionally separated at the borderbetween the outer and inner medulla by the ambient vascularsystem and by the axial heterogeneity of the epithelium alongthe descending limb.
Transport properties of thin descending limb
Osmotic equilibration along the descending limb
The tubular fluid in the descending limb is concentrated as itflows toward the tip of the renal papilla as shown by micro-puncture studies in the exposed renal papilla of hamsters [44,45], rats [46—50] and Psammomys obesus [44, 51—53]. Theoret-ically, there are two possible ways by which the luminal fluid isconcentrated along the descending limb, absorption of waterand addition of solute.
In the hamster renal papilla, Gottschalk et al [44] reportedthat sodium and attendant anions accounted for 64% and ureafor 19% of osmotic pressure of the fluid in the Henle's loop.Since the average value for tubular fluid—to—plasma (TFIP)inulin ratio was 11, 91% of the filtered water was reabsorbed inthe segments proximal to the hairpin turn of the Henle's loop.This suggests that the fluid in the hamster descending limb isequilibrated with that in the interstitium mainly by absorption ofwater. Using a sophisticated technique of puncturing the samedescending limb at two different sites, one at the hairpin turnand the other at a point 1 mm proximal to the hairpin turn,Marsh [45] was able to use a paired analysis to compare thecomposition of the tubular fluid obtained from these sites. Heshowed that at the terminal portion of the descending limb theTFIP ratios for inulin, osmolality and sodium all increased byapproximately 10%, indicating water extraction from the de-scending limb. However, he also found that a substantialamount of urea entered this segment as well.
Jamison et al [49] demonstrated that in hereditary diabetesinsipidus rats (Brattleboro strain) the rise in osmolality of thefluid in the end descending limb after administration of vaso-pressin was due to about 60% water extraction and 40%addition of non-electrolyte solutes. A subsequent study byPennell. Lacy and Jamison 1501 disclosed that 33 to 40% of the
Thin loops of Henle 567
Table 1. Transport parameters of the thin loops of hamster
Data are cited from [201, [651, and [80]. Data are expressed as means 5EM (number of experiments). Abbreviations are SDL, short—loopdescending limb; LDLu, upper portion of long—loop descending limb; tAL, thin ascending limb; Pf, osmotic water permeability; Ke, effiuxcoefficient; o-, reflection coefficient; ND, not determined.
increase in osmolality of the descending limb fluid is attributedto the solute entry, with urea being a principal solute.
The process of osmotic equilibration in the descending limbof Psammomys obesus appears to be different from those of thespecies mentioned above. In this species, water absorption isless important for an increase in osmolality of the descendinglimb fluid: de Rouffignac et al [5 1—53] demonstrated that 85% ofthe increase in osmolality was due to addition of sodium andthat 15% due to absorption of water. These micropuncturestudies indicate that there are species differences in the processof osmotic equilibration along the descending limb. In order toanalyse this process more in detail, we need to know thepermeabilities of this segment to water and solutes.
Permeability to waterMicropuncture studies in rats and hamsters mentioned above
indicate that the descending limb of these species must bepermeable to water. By the split—oil droplet technique inhamster renal papilla, Gottschalk [54, 55] observed rapidshrinking of the fluid droplet injected into the descending limb.This suggests that the segment is permeable to water. But it isperplexing that this phenomenon occurred whether the injectedfluid was hypertonic saline or distilled water. This suggests thatthe segment is permeable both to NaC1 and to water. However,Marsh and Solomon [56] using a similar method could notconfirm Gottschalk's observation. This serious discrepancymay be due to technical difficulty.
Using the in vivo microperfusion technique on inverted renalpapilla exposed from surgically exposed hamster kidney [57],Sakai, Tadokoro and Teraoka [58] demonstrated that thediffusional permeability for 3H-water was significantly higher inthe descending limb than in the ascending limb. Using in vitromicroperfusion of the rat excised papilla Morgan and Berliner[59] reported that diffusional water permeability (Pd) was highin the descending limb, 119 14 x i0 cm sec, a valuecomparable to that in the collecting tubules exposed to vaso-pressin.
Using the in vitro microperfusion of isolated renal tubules,Kokko [60, 61] found that the descending limb of the rabbit hasa high osmotic water permeability (Pf), 241.6 X l0 cm sec'(Table I). This finding was seriously criticized as a technicalartifact by Stoner and Roch.-Ramel [62], who observed that theosmotic water permeability of the rabbit descending limb wasactually extremely low if the outflow resistance of the perfusedtubule was reduced by cannulating the lumen with a glass
capillary. However, they found that an increase in perfusionpressure caused an irreversible increase in osmotic waterpermeability of the segment. On the basis of these observations,they argued that the high water permeability reported by Kokko[60, 61] may be an artifact due to high perfusion pressure. Morerecent studies by Abramow and Orci [63] and by Miwa and Imai[64], however, have confirmed Kokko's observation. The latterinvestigators could not confirm the observation of Stoner andRoch—Ramel [62] by using the same method. This segment wasso highly permeable to water that the osmolality of the perfus-ate rapidly equilibrated with that of the bathing fluid at the siteproximal to the collecting pipette. Miwa and Imai [64] furtherreported firm evidence that water permeability of this segmentis dependent on flow rate rather than on perfusion pressure; thehigher the perfusion flow rate, the higher the water permeabil-ity. One may, then, argue that water permeability of thesegment might be low at low flow rate. However, even atstop—flow the water permeability was shown to be high [64].The reason why Stoner and Roch—Ramel [62] reached a com-pletely opposite conclusion at present is unknown. The in vitromicroperfusion studies in rats and hamsters [20, 65, 66] alsoindicate that the descending limb is highly permeable to water.Imai, Hayashi, and Araki [65] compared osmotic water perme-ability between the LDL and SDL of hamsters, and found thatit was slightly higher in the LDLu (Table 1).
The mechanism of water transport in the descending limb hasnot been completely elucidated [67]. But Miwa and Imai [64]suggested that the single—file mechanism is responsible for thewater permeation. Recently, Imai [68] observed that p-chloro-mercuribenzene sulfonate (PCMBS) inhibited water movementin the hamster LDL. Since this effect of PCMBS was pre-vented by dithiothreitol, the blocking of sulfhydryl groups wasresponsible for the inhibitory effect of PCMBS on the waterchannel. These observations suggest that the main route ofwater transfer across the descending limb is the special waterchannel composed of protein molecules whose sulfhydrylgroups play an essential role in opening the channels. Recently,Whittembury et al [69] also found that PCMBS inhibited watertransport in the rat proximal tubule. On the other hand, PCMBShad no effect on the vasopressin—stimulated water flux in thehamster papillary collecting duct (Imai M and Kondo Y, unpub-lished observation), suggesting that the hormone dependent—water channel in the collecting tubule is different in nature fromthat in the more proximal segments.
568 Jmai ci a!
Permeability to sodium and chloride
By microperfusion of the descending limb of the rat papilla invitro Morgan and Berliner [59] reported that the segment waspermeable to sodium. The permeability to Na was 34 to 47 )<lO cm sec'. A similar result was found by Sakai, Tadokoroand Teraoka [58] in the hamster exposed renal papilla in vivo.These findings suggest that the descending limb of long—loopnephron is permeable to sodium. On the other hand, Kokko [60]reported that the descending limb of rabbit kidney has a lowpermeability to sodium, 1.61 x iO cm seC'. Abramow andOrci [63] reported an even much lower value, 0.17 x lO cmsec — , in the rabbit inner medullary descending limb.
Because of the difference in methodology, it is difficult todecide from these data whether there are interspecies differ-ences or internephron heterogeneity in sodium permeability ofthe descending limb. Imai et al [65, 66] examined this issuemore in detail by the in vitro microperfusion technique (Table1). In the hamster kidney, the permeability to sodium was muchhigher in the LDLu than in the SDL (45.0 5.9 vs. 4.2 2.0 xl0- cm sec'). The permeability to chloride was also higher inthe former than in the latter (4.2 0.7 vs. 1.3 0.3 x i05 cmseC'). The LDLu is 10 times more permeable to sodium thanto chloride. These observations are in good agreement with theview predicted from the morphologic observations that theLDLu is a leaky epithelium whereas the SDL is a tightepithelium.
The mechanism of sodium chloride transport across thedescending limb is unknown. From its ultrastructural appear-ance and Na+K+ ATPase content [7(1-72], one may predictthat some active transport processes exist in the LDL. It hasbeen speculated that this segment may secrete NaCl into thelumen [29, 70, 71]. However, the study of Imai et al [65, 66] didnot support this hypothesis: they showed that in the hamsterLDL there is no appreciable transmural voltage (VT), netwater flux, or net chloride flux when the composition of theperfusate is identical to that of the bathing fluid.
Permeability to urea
Morgan and Berliner [59] reported that a significant amountof urea entered descending limb when urea was added tobathing fluid, suggesting that this segment is moderately per-meable to urea. By contrast, the in vitro perfused rabbitdescending limb was shown to have a low permeability to urea,1.5 x i0 cm sec' [61]. This discrepancy may be explained,at least in part, by internephron heterogeneity and interspeciesdifference in the function of the descending limbs. The in vitroperfused hamster SDL was shown to be moderately permeableto urea, whereas the LDLu is less permeable to urea [65] (Table1). Because of morphologic similarity of the LDLL to the SDL,it is assumed that the hamster LDLL is also moderatelypermeable to urea.
It is important to note that the SDL is moderately permeableto urea. Since this segment is incorporated in the vascularbundles in some species and makes countercurrent contact withthe ascending vasa recta, urea trapped in the vascular bundle inthe inner medulla may be transferred from ascending vasa rectablood to fluid in the SDL, resulting in a urea recycling pathwaywhich returns the escaping urea to the nephron.
Potassium transport
It has been well established that the potassium recyclingtakes place in the renal medulla [52, 73—781. Although thephysiological significance of this phenomenon is unknown, anentry of potassium into the descending limb may be essential forthis phenomenon. Rocha and Kokko [79] reported that therabbit descending limb has a low permeability to potassium, 2.5x l0 cm sec. However, Tabei and Imai [80] recentlyobserved that in contrast, the LDLu of the hamster is highlypermeable to potassium: the salt permeability for KCI was 38.9
1.9 x i0 cm sec' as calculated from the amount ofpotassium added to the lumen by increasing potassium concen-tration in the bath. The permeability coefficient for 86Rb was 51
9.2 x l0 cm sec, a value slightly higher than that for 22Nain the same segment. The reflection coefficient for KC1 was 0.81
0.05, a value significantly less than unity. Since no activepotassium transport was demonstrated, the potassium secretionalong the descending limb is assumed to be mediated by passivediffusion.
Electrophysiological properties
Transmural voltage and conductance. By in vivo micropunc-ture study of the hamster renal papilla, Windhager [81] foundthat the transmural voltage (VT) of the descending limb was —3
0.3 mV, whereas Marsh and Solomon [56] did not find anyappreciable voltage. The in vitro microperfusion studies alsoshowed that there was no VT across the descending limb ofrabbits [60, 661, rats [66] and hamsters [65, 66] when thecomposition of the perfusate and the bathing fluids were iden-tical.
On the basis of morphologic distinctions, it is possible thatthere are species differences and inter- and intranephron heter-ogeneity in the transmural electrical resistance of the descend-ing limb. However, the information is very limited at present.Abramow and Orci [63] reported that the transmural electricalresistance of the LDLL of rabbits was high exceeding 700 Clcm2, a value typical for tight epithelia.
Permselectivity. By measuring the deflection of VT when theconcentration of NaC1 and KC1 of the bathing fluid was varied,Imai [66] examined permeability for sodium and potassiumrelative to chloride (PNajcj, P}cJc,) in the descending limb ofrabbit, rat and hamster. It was found that there are considerablespecies differences and internephron heterogeneity in theseparameters (Table 2). In hamsters and rats the LDLu had highvalues for Pjcj and PKJcI, whereas the SDL had low values forthese parameters. By contrast, in rabbits such internephronheterogeneity was less, with these parameters all being low.Although actual permeability values were not determined forrats and rabbits, these findings suggest that the LDLu of rat aswell as hamster is highly permeable to sodium and to potas-sium, whereas the SDL is less permeable to these ions, andboth LDLu and SDL of the rabbit have low permeabilities tothese ions.
Recently, Tabei and Imai [82] measured the permselectivityof the hamster LDLu by observing the voltage deflection duringionic replacement of the bathing fluid, They found that thissegment is highly permeable to cations, with the sequence ofpermeability being K > Rb > Li > NH4 = CsNa>>ClBrNO3>I > acetate. Since this segment is also
highly permeable to water, possibly having single—file waterchannels, it is tempting to speculate that the water channelsmay be cation selective like the gramicidin channel [83, 84]. Thehypothesis has been tested by observing effects of PCMBS onboth diffusion potential caused by an NaC1 concentration gra-dient and the streaming potential induced by a urea concentra-tion gradient [68]. Since PCMBS inhibited both parametersproportionally, it is possible that the water channels in hamsterLDLu have cation permselectivity. Of course, this does notnecessarily reject the view that paracellular pathway is alsocation permselective.
Transport profiles along the descending limbWe have now additional information about transport param-
eters of the descending limbs of Henle's loop as noted above.Simple knowledge of the transport parameters is not necessarilysufficient to understand phenomena taking place in vivo. This istrue especially for understanding solute concentration gradientsalong the renal medulla since the ambient solute concentrationchanges along its axis. In this section, we discuss the solute andwater transport profiles along the descending limb based mainlyon a simulation study which incorporates morphologic andfunctional heterogeneity.
Concentration processes along the descending limbIt has been controversial whether absorption of water or
addition of solute mainly contributes to the osmotic equilibra-tion of the luminal fluid along the descending limb. On the basisof a low urea permeability of the rabbit descending limb, Kokko[611 suggested that entry of urea into this limb may be minimal.However, using parameters reported by Kokko [61], Pennell etal [15] calculated that about 33 to 40% of osmolality in thedescending limb was accounted for by the entry of urea,assuming that concentration of solutes in the interstitiumchanges linearly.
Based on the observation that there are considerableinternephron heterogeneity and species differences in the func-tion of the descending limb, Taniguchi, Tabei and Imai [85]recently conducted a computer analysis to simulate the trans-port profile along the descending limb by applying phenomeno-
LDL/SDLI LDLL I tAL
Length of thin loop, mm
Fig. 2. Volumetric and solute mass flow rates along the thin loopsegments of hamster kidney simulated on the assumption that soluteconcentrations of medullary interstitium increase linearly. It was as-sumed that concentrations of Na, K and urea change from 150 m atthe cortico-medullary junction to 350 m at the papillary tip, from 5 to50 m, and from 5 to 300 m, respectively. Solutes and water wereassumed to be transported by passive mechanisms. For solute move-ment, both electrical driving force by diffusion potential and solventdrag were also taken into consideration. All transport parameters werederived from experiments in hamsters. Solid lines represent thelong—loop nephron, broken lines the short—loop nephron. The data onthe descending limb of long—loop nephron are cited from Taniguchi et a![85]. Abbreviations are: LDL, upper portion of the long—loop descend-ing limb; LDLL, lower portion of the long—loop descending limb; SDL,short—loop descending limb; and tAL, thin ascending limb.
logical coefficients for solute and water transport in the ham-ster. Although in our original analysis [85] major attention wasfocused on the profiles along the long—loop nephron, in thiscommunication we add some data comparing the profiles of thelong—loop nephron with those of the short—loop nephron. Sev-eral assumptions were necessary for simplifying the analysis.The mathematical model developed by Taniguchi et al [85] wasvery similar in principle to that reported by Pennell, Lacy andJamison [50] and Jamison et al [73]. Mass balance equations forsolutes and water were solved by assuming that concentrationof solutes in the interstitium increased linearly along thecortico-medullary axis, with NaCI, KC1 and urea increasingfrom 150 to 350, 5 to 50, and 5 to 300 m, respectively. Theelectrical driving force due to diffusion potentials and solventdrag were considered to contribute to solute movements. Fig. 2summarizes computed profiles for mass flow rates of water,Nat, K and urea along the descending limb of either short— orlong—nephron.
Water reabsorption. Since the descending limb of Henle ishighly permeable to water, one may assume that water absorp-tion mainly accounts for the osmotic equilibration of the luminalfluid. As shown in Figure 2, water absorption is much greater inthe long—loop than in the short—loop nephron. This is in part dueto the fact that the descending limb of the long—loop is lesspermeable to urea than that of the short—loop. In addition, thehigh permeability for sodium is also responsible for the greaterwater absorption in the long—loop nephron because sodium,
Table 2. Internephron and interspecies differences of electricalpermeabilities for Na and K relative to Cl
Ic St
a
o-1) ca -EE6EE>
zData are expressed as means SEM (number of experiments).
Abbreviations are same as in Table 1.
2 3 4 5 6 7 8 9 9.8
570 !mai et a!
rather than causing an increase in osmolality of the luminalfluid, diffuses out of the lumen. Water absorption is more rapidin the outer medulla than in the inner medulla. Under standardconditions simulating micropuncture studies, about 75% and40% of the entered water is reabsorbed along the descendinglimb of the long—loop and of the short—loop nephron, respec-tively.
Sodium transport. Based on the observation that the perme-ability to sodium was very high in the LDL, it has beenspeculated that net sodium entry could occur into the descend-ing limb [65, 66]. However, it is surprising to note that the resultof the simulation study was completely opposite to this specu-lation: a considerable amount of sodium was reabsorbed alongthe long—loop descending limb (Fig. 2). Both the high perme-ability to sodium and the steep cortico-medullary osmoticgradient are essential for this phenomenon. Under the sameosmotic gradient, sodium reabsorption is much less in theshort—loop descending limb where the permeability to sodium isvery low (Fig. 2). Increases in concentration gradient of ureawere associated with increases in sodium reabsorption in theouter medulla. In the LDL, net sodium entry may occur, butonly in the very proximal portion. By absorption of water,luminal sodium concentration increases, leading to outwarddiffusion of sodium. In spite of reabsorption, the sodiumconcentration remains higher in the lumen than in the intersti-tium throughout the renal medulla, except in the very earlyportion. Under standard conditions, sodium concentration inthe lumen of the end descending limb was 15 mEq/liter higherthan in the interstitium.
This theoretical analysis is interesting because it tells us thatwe cannot predict sodium entry into the descending limb simplybased on the high permeability to sodium. However, theamount of sodium reabsorption calculated here appears to bemore than that observed by micropuncture studies. Jamison,Buerkert and Lacy [49] reported that in Brattleboro rats therewas little or no sodium entry into the descending limb. Frac-tional delivery of sodium at the hairpin turn in Brattleboro ratswas in the range from 30 to 50% of filtered load, which wasunaffected by administration of vasopressin [47, 50]. Thus, it ispossible that our model slightly overestimates sodium reabsorp-tion along the descending limb. Possible sources for this over-estimation deserve to be considered.
First, sodium permeability used for the calculation might betoo high because the experimental data were obtained from thethickest LDL (Type ha). In addition, it is also possible thatsodium permeability decreases as a function of the length of theLDLu. Second, it is highly likely that composition of theinterstitium around the LDLu is different from that around theSDL because of the differences in tubulo-vascular architecture.Since the LDL is adjacent to the thick ascending limb, it ispossible that to sodium concentration in ambient interstitium ishigher than in the region of the vascular bundles. To examinethis possibility, Taniguchi, Tabei and Imai [85] used the modelto see whether net sodium entry could be demonstrated byreplacing urea with sodium chloride in the interstitium of theouter medulla (Fig. 3). Although this maneuver reduced sodiumreabsorption, no net addition of sodium was observed evenwhen almost all urea was replaced by sodium chloride.
The results of the in vitro studies [65, 66, 80, 82] andmathematical analysis [85] from our laboratory are in good
agreement with the observations in a series of micropuncturestudies by Elalouf, Roinel and de Rouffignac [86—88], whoexamined effects of vasopressin [86], calcitonin [87], glucagon[88], and PTH [88] on water and electrolyte transport in thejuxtamedullary Henle's loop in Brattleboro rats deprived of anendogenous source of these peptide hormones. Each of thesehormones, except PTH, is known to stimulate NaCl transport inthe thick ascending limb [86-881. The conclusions from thesestudies are: 1) there is sodium reabsorption along the descend-ing limb in hormone—deprived rats; 2) the amount of sodiumreabsorption is amplified when removal of water along thedescending limb is enhanced by administration of vasopressin[86] or calcitonin [871; and 3) addition of sodium occurs whensodium transport across the thick ascending limb is stimulatedin the absence of an effect on the collecting tubules, as observedin the case of glucagon [88], where water removal from thedescending limb is prevented.
In conclusion, the orientation and magnitude of sodiumtransport across the long—loop descending limb are criticallydependent on the transmural gradients of sodium and osmolal-
1.5[1.0
0E
5)C',
0(0CC'C',
E
0.5z
0
Length of descending limb, mm
Fig. 3. Effect of changes in sodium concentration in the interstitium ofthe outer medulla on Na+ mass flow rate along the long—loop descend-ing limb. Sodium concentration at the border between outer and innermedulla was increased with a step of 10 m by replacing withequiosmolal urea. Condition I is the same as in the data shown in Figure2. A little amount of net sodium reabsorption was observed only whenurea concentration was reduced to 7.8 msi (I). [NaCI]1 and [Urea]1denote concentration of the respective solute in the interstitium at thejunction between outer and inner medulla, respectively.
Thin loops of Henle 571
ity. By contrast, sodium transport across the SDL is minimaland is affected little by these parameters.
Urea transport. As already discussed, a considerable amountof urea enters the descending limb even if the permeability tourea is low [151. If we assume that the permeability to urea inthe LDLL is similar to that in the SDL, a large amount of ureais calculated to enter the descending limb of the long—loopnephron (Fig. 2). It should be noted that an increase incortico-medullary urea gradient actually diminishes the entry ofurea. This apparent paradoxical phenomenon can be explainedby the rapid osmotic equilibration which diminishes thetransmural urea concentration gradient. On the other hand, it isof interest to note that an increase in urea gradient causesfurther increases in urea entry in the short—loop nephron (datanot shown). These observations, in combination with the archi-tectural organization of the SDL with vascular bundles, are infavor of the view that the short—loop nephron is more importantfor urea recycling.
Potassium recyclingAs noted in the case of sodium transport, a simple consider-
ation of permeability value is not sufficient to predict thetransport profile of any solute in question. As shown in Figure2, the simulation study demonstrates that a considerableamount of potassium enters the long—loop descending limbunder the condition simulating micropuncture studies. There-fore, the permeability coefficient for potassium determined inthe hamster descending limb support the hypothesis that thedescending limb of Henle's loop is the major site of potassiumrecycling. Although active potassium secretion has been dem-onstrated in the rabbit proximal straight tubule (89, 90, Miwa T,Tabei K and Imai M, unpublished) the amount of potassiumsecreted in this segment is not sufficient to account for theamount of potassium observed by micropuncture [90, 91].Taniguchi, Tabei and Imal [85] found by the mathematicalmodel that the mass flow rate of potassium at the end descend-ing limb changed very little when the amount of potassiumdelivered to the descending limb varied to a large extent,suggesting that the proximal tubules may not play a significantrole in the recycling of potassium.
Transport properties of thin ascending limbThe function of the thin ascending limb of Henle's loop (tAL)
has been a matter of considerable discussion. The major dis-crepancy concerns whether active transport exists in this seg-ment. In view of morphologic similarity of the tAL amongvarious species, the divergent results in the functional studiesmight be accounted for by methodological rather than speciesdifferences.
Dilution of tubular fluid along the thin ascending limbGottschalk and Mylle [92] reported that in the hamster the
samples obtained at the same level in the medulla from thebends of loop of Henle, collecting ducts, and vasa recta wereequally hypertonic. Using the technique developed by Sakai,Jamison and Berliner [57] for exposing renal papilla fromsurgically excised kidney, Jamison, Bennett and Berliner [47]demonstrated in the rats that osmolality of the tubular fluidcollected from the tAL was about 120 mmoles/kg H20 lessconcentrated than that from the descending limb at comparable
levels in the medulla. Sakai, Tadokoro and Teraoka [58], usingthe same technique, obtained a similar result in the hamster.Jamison, Bennett and Berliner [47] demonstrated further thatthe lower osmolality of the fluid in the ascending limb is almostentirely accounted for by a lower sodium concentration. Asubsequent study by Jamison [46] demonstrated that the frac-tion of filtered sodium was lower in the thin ascending limb thanin the descending limb. These findings indicate that fluid in thethin ascending limb is relatively diluted by reabsorption ofsodium.
By paired puncture at the bend of loop and at one mm alongthe ascending limb, Marsh [45] clearly demonstrated that astubular fluid flowed upward there was a fall in sodium concen-tration, and no change in inulin concentration. By in vitromicroperfusion of rabbit tAL, Imai and Kokko [18] demon-strated that, under a special experimental condition in whichtubules were perfused with serum ultrafiltrate, the osmolality ofwhich was increased by 163 mr'.'i NaCI while bathing withserum, the osmolality of which was made equiosmolar byadding 300 mivi urea, osmolality of the collected fluid clearlydecreased as a function of the perfused length. This observationsuggests that luminal fluid could be diluted by diffusion of NaCIin excess of entry of urea by a purely passive mechanism.
Permeability to water
The early split—oil droplet study by Gottschalk [54, 55]suggested that the hamster tAL is impermeable to water. Thisobservation was not supported by Marsh and Solomon [56],who showed that injection of raffinose solution caused anincrease in volume of droplets. Using the in vitro microperfu-sion on rats kidney slices, Morgan and Berliner [59] reportedthat diffusional water permeability was low (50 x l0 cmsec). This was supported by the observations by Sakai,Tadokoro and Teraoka [58] in the in vivo perfused tAL ofexposed hamster papilla. Marsh [45] observed in hamsters thatthere was no change in inulin concentration between pairedsamples obtained at the bend of the loop and a more distal siteof the tAL. In vitro microperfusion studies showed that osmoticwater permeability of the tAL of rabbits [18], rats [20], andhamsters [20] was extremely low, and not significantly differentfrom zero. Diffusional water permeability of rabbit tAL wasalso reported to be very low, being 45.3 x l0 cm sec' [18].Although the value was increased to 52.7 x l0 cm sec' by anaddition of 0.2 mU/mi vasopressin, this was interpreted not tohave any physiological significance. Taken together these datashow that the tAL is virtually impermeable to water.
Permeability to sodium and chloride
The early split—oil droplet studies [54—56] suggested that thehamster tAL is highly permeable to sodium chloride: thecomposition of the solution injected into the tubular lumenrapidly equilibrated with that of the adjacent vasa recta or finalurine without significant change in the volume of luminal fluid.
The in vitro microperfusion studies of the tAL in the excisedpapilla of the rat kidney [59, 93, 94] have led to a somewhatdifferent conclusion. When the osmolality of the bathing solu-tion was increased by adding NaCl, osmolality of the collectedfluid changed very little in the absence of change in inulinconcentration, suggesting that this segment has a low perme-ability to NaCI as well as to water [59]. This was confirmed by
572 Imai et a!
the measurement of sodium permeability with 24Na in thesimilar preparation (7 x lO- cm sec') [93]. It was shown thatthe permeability to sodium was less in the tAL than in thedescending limb [59, 93, 941, On the other hand, Sakai,Tadokoro and Teraoka [58] found by a similar technique that inthe hamster both descending and ascending limbs were equallypermeable to 22Na.
In vitro microperfusion studies demonstrated that the tAL ofrabbits [18, 19], rats [20], and hamsters [20, 97] were highlypermeable to sodium and to chloride. The permeability tosodium of the rabbit tAL was about 25.5 x l0— cm sec', avalue that is five times higher than that in the proximalconvoluted tubule. The permeabilities to sodium of the tAL inhamsters and rats were 79.6 and 87.6 X l0— cm sec,respectively. The permeability to chloride was much greaterthan to sodium: the values were 117.0, 183.7 and 196.0 X i0cm sec', in rabbits, rats and hamsters, respectively. Since thissegment is impermeable to water, salt permeability for NaC1can be estimated by measuring osmolality of the collected fluidwhen a transmural NaC1 gradient is imposed. The salt perme-ability to NaCl was shown to be 82.2 x l0 cm sec', a valuewhich was very close to the permeability to 22Na [20]. Thissuggests that the permeability to sodium rather than to chloridesomehow becomes a rate limiting factor for the transmuralmovement of NaCl.
At any rate, the results of the in vitro microperfusion studieson isolated tubules strongly suggest that the tAL is highlypermeable to NaCl, The reason for discrepancy with the datafrom other laboratories with different techniques is unknown.However, the possibility cannot be ruled out that the transportproperties may differ between the upper and lower portion ofthe tAL.
Permeability to ureaThe permeability of the tAL to urea is of ultimate importance
in the passive equilibration model proposed by Kokko andRector [17]. Morgan and Berliner [59] demonstrated that theentry of sodium chloride into the rat tAL was less than the entryof urea when transmural osmotic gradients were imposed byadding the each solute to the bathing medium, implying that thesegment is more permeable to urea than to sodium. Thesetransport properties are completely opposite to those predictedfrom the passive models [16, 17].
On the other hand, the direct measurement of urea perme-ability in isolated perfused tAL [18, 20] provided a valuefavorable for the model, that is, the segment is less permeableto urea than to NaCl. The permeabilities to urea of the tALwere 6.7, 22.8 and 18.5 x iO cm sec' in rabbits, rats andhamsters, respectively.
Electrophysiological propertiesNo detailed electrophysiological study has ever been per-
formed in the tAL. Only transmural voltage and permselectivityhave been reported.
Transmural voltage. The earlier micropuncture studies inhamster kidney [56, 81] showed that the transmural voltage (VT)of the tAL was ranged from —9 to —11 mV. Windhager [81]suggested active transport for the source of the VT, whereasMarsh and Solomon [56] attributed the origin of the VT to astreaming potential generated by volume flow. Because of a
variety of methodologic problems in earlier studies, the resultsof these studies might not be reliable.
To avoid the possible interference with liquid junction poten-tial between the reference electrode and the composition of therenal medullary tissue, Marsh and Martin [95] measured thevoltage difference in vivo between free—flow tAL fluid andascending vasa recta in hamster kidneys. They found that theVT of the tAL was about 2 mV oriented positive in the lumen.Since this lumen positive VT was abolished by intraluminaladministration of either furosem-ide or ouabain, they concludedthat the lumen positive VT represents active, electrogenicchloride transport. In addition, by measuring diffusion potentialin the presence of NaCI gradient, they confirmed that thissegment was two times more permeable to chloride than tosodium. Hogg and Kokko [96] also found the lumen positive VTof 2 mV in the rat tAL studied in vivo. However, they arguedthat the lumen positive VT is caused exclusively by preferentialpermeation of chloride, because they observed that the polarityof the VT was reversed when the transmural chloride gradientwas reversed.
In vitro microperfusion of the tAL of rabbits [18, 19], rats [20]and hamsters [20, 97] failed to demonstrate any appreciablevoltage when the composition of the perfusate was identical tothat of the bathing fluid. These findings, however, do notnecessarily exclude the possibility that the in vitro preparationmay lack some humoral factors necessary for generation of theVT. Vasopressin is potentially one such factor, since thishormone has been shown to stimulate chloride transport acrossthe medullary thick ascending limb of rats [98] and mice[98—100]. Imai and Kusano [97] examined in the hamster andmouse tAL whether vasopressin generates a VT or stimulateschloride transport. However, no appreciable VT was observedin the tAL in the presence of vasopressin. They further dem-onstrated that vasopressin or dibutyryl cyclic AMP caused littleor no change in chloride fluxes. They also confirmed thatvasopressin does not affect the permselectivity revealed by thediffusion potential induced by an NaC1 gradient. Therefore, theabsence of vasopressin is not prerequisite for the failure todemonstrate in vitro the VT across the tAL.
Permselectivity. Using the in vitro perfused tAL, Imai et al[18, 19, 20, 97, 101] reported that, when a transmural NaC1gradient was imposed, a diffusion potential was generated withan orientation compatible with the preferential permeation forchloride. From the deflection of the VT at a given transmuralNaCl gradient, we can calculate the relative transference num-ber or electrical permeability ratio (PCIJNa). The values for PCain the tAL of rabbits, rats and hamsters were 3.5, 2.35 and 2.17,respectively [20, 101]. The value in the hamster is in goodagreement with that observed by the in vivo micropuncturestudy [95].
From the deflection of VT which occurred when sodiumchloride in the bathing fluid was replaced by various salts,electrical permeabilities for various ions relative to sodium canbe calculated with Goldman's constant field equation. Theresults obtained in the tAL of rabbits [101], rats [20], andhamsters [20] are summarized in Figure 4. It is clear from thisfigure that this segment is permselective for halides includingCl, Br, 1, and SCN rather than for anions in general.
Active solute transport. Almost all the computer analyseshave suggested that the existence of active salt transport in theentire ascending limb is the most favorable for generating asteep osmotic gradient along the cortico-medullary axis. How-ever, in spite of extensive efforts, no one has ever succeeded indemonstrating unequivocal evidence for the existence of activesolute transport across the tAL. Marsh and Azen [1021 reportedthat in hamsters, under either antidiuretic or saline diureticcondition, Na concentration in the tubular fluid obtained at thebend of the loop or at the site of the tAL 1 mm distal to the bendwas not different from that in the adjacent vasa recta. But whenflow rate was slowed down by aspirating tubular fluid at a siteproximal to that of the collection, they found that the sodiumconcentration in the fluid of the tAL fell about 50 mEq/literbelow that in the adjacent plasma. Taken together with theobservation by Marsh and Martin [95] mentioned above, theseinvestigators argued that an electrogenic active chloride trans-port occurs in the tAL. Since Na-K ATPase activity in thissegment has been shown to be unmeasurably low [103], it isdifficult to understand why ouabain inhibited the VT even byintraluminal administration. In addition, it is possible that theefficiency of active chloride transport, if present at all, must bevery limited by a large amount of back leak, because thissegment is highly permeable to chloride.
On the other hand, Imai and Kokko [18] failed to demonstrateany net transport of solutes including sodium, chloride and ureaacross the rabbit tAL perfused in vitro when the perfusate andthe bathing fluid were identical in composition. This observa-tion is in favor of the view that there is little or no active solutetransport in this segment.
Evidence for interaction with the membrane in chloridetransport. Since the tAL is extraordinarily highly permeable tochloride and has a unique characteristic of halide permselec-tivity, it is possible that there is a special mechanism whichfacilitates the transmural movement of halides. To examine thispossibility, Imai and Kokko [19] performed a detailed study ofthe transport kinetics of sodium and chloride in the rabbit tAL.The flux ratio analysis proposed by Ussing [104] is expected to
provide a means to differentiate a simple passive mechanismfrom other more complicated passive mechanisms. If the x ionis transported across a membrane in the presence of thetransmembrane voltage of i.V mV, the ratio of the unidirec-tional flux from lumen—to—bath (JXLB) over the flux frombath—to—lumen (JXBL) can be expressed as:
(JX/JX) = (CXL/CXB) exp(zFzV/RT) (1)
where CXL and CB are concentration of x in the lumen and thebath, respectively, and F, z, R and T have their usual meanings.Bidirectional fluxes for 22Na and 36C1 were measured underthree different conditions in which the transmural voltage wasvaried by inducing diffusion potentials. The results shown inFigure 5 indicate that the flux ratios for Na1 are distributed onthe line predicted from the equation 1, implying that sodiumtransport across the tAL is mediated entirely by simple passivediffusion. This was supported by another observation that thelumen—to-bath flux of sodium was a linear function of sodiumconcentration in the luminal fluid [19]. Figure 5 also shows thatthe flux ratios for Cl are not those predicted by equation 1.Although this finding suggests that chloride transport is medi-ated by some mechanism other than simple passive diffusion, itis impossible to identify the exact mechanism. A single—filemechanism is excluded, however, because the slope is less than1.0.
When analyzed by subtracting the predicted simple passivecomponent, the lumen—to-bath 36C1 flux was shown to reach acertain maximal level as chloride concentration was increased[19]. The lumen—to-bath 36C1 flux is competitively inhibited bybromide [19]. Taken together with the observation that the tALis permselective for halides, the membrane might have trans-porters with binding sites for halides. Such transporters couldbe either carriers or channels with properties other than thesingle—file mechanism. It remains uncertain whether such trans-porters are located on cell membranes or paracellular junctions.
Although the symmetrical characteristics of the diffusionpotential [20] favor the view that the halogen—selective pathwayis through the tight junction, we cannot rule out the possibilitythat there are halogen conductive pathways in the luminal and
Thin loops of Henle 573
zci-ci-
4
3
2
0C1 HCO3 AcetateCyclamate
Mechanisms of sodium and chloride transport in tAL
Fig. 4. Electrical permeabilities for variousions relative to Na in the thin ascendinglimb of rabbits (LI), rats () and hamsters(. Figure has been constructed according to
Br SCN— K Li ChoIine the data from [20, 1011.
574
exp(ZFW/RT)
Imai et al
Fig. 5. Flux ratio analysis for Na and Ct transport in the rabbit thinascending limb. Bidirectional fluxes (lumen—to—bath and bath—to—lu-men) were determined for Na (—•—) and Cl- (— .0—.) under threedifferent conditions, where transmural voltage (V) was varied byvarying transmural NaCl gradients. Unidirectional fluxes were normal-ized by the concentration of respective ions. Regression lines for Naand Cl are: y = 0.98x — 0.01 (r = 0.93) andy = 0,42x + 0.54 (r = 0.81),respectively. The line for Na was not different from the line of identity(y = x), whereas the line for C1 was clearly deviated from the latter.From [19] with permission.
basolateral membranes in series. A more detailed characteriza-tion of the transporters is now under extensive investigation inour laboratory. Although Marsh [105] has proposed the possibleexistence of a Cl/urea antiport mechanism in the tAL, ourpreliminary study in hamsters failed to support such hypothe-sis.
Transport profiles along thin ascending limbThe transport properties of the tAL demonstrated by the in
vitro microperfusion technique of isolated nephron fragmentsare quite compatible with those predicted by the model ofKokko and Rector [17]. As already mentioned, such transportproperties allow a transmural osmotic gradient to be generatedin the absence of active solute transport under specific experi-mental conditions [18]. In order for the passive model to beoperative also in vivo, driving forces favorable for passiveoutward diffusion of sodium chloride must exist. Johnston et al[1061 found in antidiuretic rats that the mean concentration ofsodium in the descending limb fluid was 344 mEqlliter, a valueabout 60 mEq/liter higher than that in the adjacent vasa rectaplasma. When correction was made for the plasma proteinconcentration in vasa recta, the calculated transmural gradientwas 40 mEq/liter. Hogg and Kokko [96] also found in the ratthat chloride concentration in the loop fluid was higher by 35 to40 mEqlliter than that of adjacent vasa recta fluid. A similarresult was also reported by Gelbart et al [1071. The simulationstudy by Taniguchi, Tabei and Imai [85] predicted similarresults, although the gradient was slightly lower in the hamsterand greater in the rabbit.
IAL CNW PCD
C—2 C—3
'I,
H2OUrea
Urea+ Urea
NaCI
M-I M-II
+ + (r NoCI>(TUre)
0
Purea + +
Fig. 6. Simple three compartment model (left) and assignment of eachcompartment to the structures of the inner medulla (right). C-i, C-2 andC-3 represent three compartments, where initial solute compositionsare indicated in the bottom. Membrane I (M-I) is impermeable to water,highly permeable to NaC1 and moderately permeable to urea. Mem-brane II (M-II) is highly permeable to water and moderately permeableto urea, but impermeable to NaCl. The reflection coefficient of thismembrane is higher to NaCl than to urea. Abbreviations are: CNW,capillary networks; VB, vascular bundle; DLH, descending limb ofHenle; tAL, thin ascending limb; PCD, papillary collecting duct.
Although these results are favorable for the model of Kokkoand Rector [17], it is uncertain whether continuous dilution ofintraluminal fluid can be established throughout the thin ascend-ing limb. In order to examine this issue, we simulated thetransport profiles along the tAL by a model similar to that usedfor the descending limb. We assumed again that the concentra-tion of NaCl, KC1 and urea in the interstitium change linearly.The transport profiles were simulated for both hamsters andrats by using actually measured parameters. The results areshown in Figure 2. In both species, the entry of urea is observedonly in the very initial portion of the tAL. Thereafter urea isreabsorbed along the tAL (Fig. 2). Continuous entry of ureaalong the tAL does not occur, because so much urea hasalready entered the descending limb and because the perme-ability to urea of the tAL is relatively high. Therefore, we haveto admit that the countercurrent multiplication cannot be oper-ated by the simple passive mechanism, at least in the mannerproposed by Kokko and Rector [17].
Working hypothesis on countercurrent system bytubulo-vascular complex
Although the transport properties of the thin loop segmentshave been clarified to a certain extent, it is still far fromcompletely clear how the thin segments participate in the urineconcentrating mechanism. Since two solute (NaCl vs. urea)models are still attractive in the sense of the economy ofenergy, we need to seek alternative hypotheses which do notrequire active salt transport in the inner medulla. In this lastsection, we describe simple principles of our idea about the roleof the thin loop segments in the mechanism of urine concentra-tion in the hope of assisting further detailed formulation for
computer analyses. Our working hypothesis is depicted sche-matically in Figures 6 and 7.
Single effect by passive diffusion of solutes in threecompartments
Before discussing the countercurrent multiplication system,let us consider a special form of "single effect" in threecompartments which are separated by two membranes withunique transport properties (Fig. 6). The membrane I (M-I) isimpermeable to water, but highly permeable to NaCI andmoderately permeable to urea. The membrane II (M-II) ishighly permeable to water and moderately permeable to urea,but impermeable to NaC1. The reflection coefficient of thismembrane is higher for NaC1 than for urea (o- ij NaCI> 011 Urea).Initially compartment 3 (C-3) contains a pure urea solution,whereas both compartments 1 and 2 (C-l,C-2) contain anequiosmolal solution consisting of NaC1 and urea. Although thenominal osmolality (that is, osmolality determined by freezingpoint measurements) of the solutions is identical in C-2 and C-3,the effective osmolality of the solution in C-2 is higher than thatin C-3, because II NaCI> fl Urea Therefore, a volumetric fluxoccurs from C-3 to C-2, resulting in concentration of urea in C-3and dilution of NaC1 and urea in C-2. The latter process createsa concentration difference which favors diffusion of NaCl andurea from C-i to C-2, resulting in dilution of compartment 1without change in volume (the single effect). Diffusion of NaCIand urea into C-2 increases osmolality of this compartment,further promoting water absorption from C-3. Net results ofthese processes are: 1) dilution of C-l solutes without change involume; 2) increase in solute content of C-2 with an increase involume; and 3) concentration of C-3 solutes with a decrease involume. Under this condition we must postulate the followingparadoxical relations:
H 112
(oi H)2(o-ll 11)3
H3 H(3)
(4)
where H1, 112 and 113 denote nominal osmolality of C-i, C-2, andC-3, respectively. (a- H) means effective osmolality acrossmembrane II. This means that the solution in C-2 is nominallyhypo-osmotic to that in C-i, effective osmolality in C-2 is higherthan in C-3.
The above description, however, is not accurate, because weneglected possible diffusion of urea from C-3 to C-2. If the ureapermeability of membrane II is high, C-3 cannot be concen-trated. Therefore, the urea permeability of membrane II shouldnot be too high. At any rate, the system described above canperform osmotic work provided that solutions with differentsolute composition are supplied. Therefore, if such a system isincorporated into a counter—flow system, it will provide a singleeffect for the multiplication system.
This model appears to be similar to that proposed byStephenson [16], but the assignment of actual renal tubules foreach compartment is different. We assign compartments C-2 tothe tAL, C-2 to the capillary networks and C-3 to the collectingduct, respectively (Fig. 6). Thus, the descending limb of Henleis not included as one limb of a multiplier. Rather, we assumethe descending limb constitutes a countercurrent exchangerpaired with ascending vasa recta.
Complex counterfiow system in the renal medulla
Alhough we have not yet performed a computer analysis onthe whole system, our working hypothesis on the countercur-rent system with tubulo-vascular complexes is depicted sche-matically in Figure 7. The major points that we wish to addressin this hypothesis are threefold. First, we assume that renalvessels make countercurrent pairs with renal tubules and thatthey function not only as a countercurrent exchanger, but also
(2) as a countercurrent multiplier. Five different pairs are illus-
Renal pelvis
576 Imai et a!
trated in Figure 7. The first counterfiow pair is the thickascending limb and descending vasa recta (DVR) (No. 1 in Fig.7). For illustrating purpose, the DVR are represented by asingle capillary. This pair of the thick ascending Iimb-DVRconstitutes the countercurrent multiplier in the classical form,with the single effect being active NaC1 transport in the thickascending limb, generating the NaC1 gradient in the outermedulla. The LDLu cannot serve as the descending partner ofthe thick ascending limb, because NaC1 pumped out from thethick ascending limb cannot enter the LDLu as already ana-lyzed. The second counterfiow pair is the combination of SDLand ascending vasa recta (AVR) (No. 2 in Fig. 7) whichconstitutes the peripheral ring of the vascular bundle in somespecies like rats and mice. This pair may operate as a counter-current exchanger for urea as well as water since the SDL ismoderately permeable to urea. The third counterfiow pairconsists of the LDL and AVR (No. 3 in Fig. 7). This pair mayoperate as a countercurrent exchanger, causing water absorp-tion and sodium reabsorption from the LDL. The fourth pair iscomposed of the LDLL and AVR (No. 4 in Fig. 7).
The fifth pair is the combination of the tAL, AVR andpapillary collecting duct (PCD), which correspond to threecompartments mentioned above (No. 5, 6, in Fig. 7). This pairplays the most important role as a countercurrent multiplicationsystem generating the axial osmotic gradient in the innermedula. The co-existence of the collecting duct (No. 6 in Fig. 7)may be important both for supplying urea to the system and forallowing the urine to be concentrated at the same time asalready discussed. Since the tubulo-vascular organization in theinner medulla is not distinct, actual combination of these pairsmay be less obvious. But the tAL must be separated from theLDLL. If we allow the ascending and descending vasa recta tobe commingled, the structure of the model becomes similar toStephenson's model [161. For the moment we prefer to assumethat the fourth pair is separated from the fifth pair. The singleeffect leading to multiplication of urea through these twocombined countercurrent systems has been already discussed.Although the permeability to urea in the tAL is lower than thepermeability to NaC1, the absolute value for urea permeabilityis still high. As already shown by the computer simulation, ureamust diffuse out of the tAL except in the initial portion. Thesimulation study of the LDLL also showed that urea enters thelumen of this segment. The energy source for supplying highurea concentration at the hairpin turn is, of course, the activeNaCI transport in the thick ascending limb as predicted in thepassive models [16, 17].
The second point of our working hypothesis concerns theroutes by which urea is supplied to these multipliers. For anefficient operation of the countercurrent multiplication of theinner medulla, urea must be added only to the tip of the loop.Indeed urea is primarily reabsorbed by the papillary collectingduct in the presence of ADH. Moreover, it is relevant to notethat the existence of urea in urine bathing the renal pelvis hasbeen shown by many investigators [108—111] to be important ingenerating a concentrated urine. However, Marsh and Martin[112] reported that in antidiuretic hamsters there was no evi-dence for reduction in mass flow rate of urea or of other solutebetween urine in the collecting duct at the papillary tip andurine downstream in the ureter, which argues against urearecycling from pelvis to papillary tip. On the other hand, they
found that substantial amount of urea was reabsorbed at theterminal portion of the collecting duct. These latter findings areless attractive for our model but do not necessarily deny it.There are alternative or additional possible mechanisms whichfavor the urea entry into the tip of loops. If the permeability tourea of the papillary collecting duct increases along the axismore urea will enter in the area close to the papillary tip.Recently, Kondo in our laboratory made preliminary observa-tions that in the rat papillary collecting duct perfused in vitro,the vasopressin—stimulated urea permeability was higher in thedistal portion than in the proximal portion (unpublished). TheHenle's loops make their turns at various levels in the innermedulla. The longest loops are distributed around the collectingducts, and as the loops become shorter as they are displacedfrom the collecting duct. When the countercurrent system ofthe longest pair generates a urea gradient, the tip of the nextpair will be dipped in high urea environment. If this eventoccurs successively in chain, urea gradient may be generated inentire papilla.
The third point we wish to address is that in this model theshort—loop nephron and the long—loop nephron play separateroles: the former is more important for recycling of urea as anexchanger whereas the latter operates as a multiplier.
Although the working hypothesis discussed above is highlyspeculative and has not been subjected to quantitative analysis,it is a possible mechanism for the efficient operation of theconcentrating mechanism.
AcknowledgmentsWe would like to express our thanks to Dr. Rex L. Jamison for his
valuable suggestions in analyzing our experimental data. We also thankDrs. Christian de Rouffignac and Lise Bankir for kindly providing uswith some of their manuscripts under submission or in press. Weappreciate Drs. Koji Yoshitomi and Yoshiaki Kondo for their activediscussion of this review. Secretarial work by Miss Man Wada wasvery helpful. Our data quoted in this review have been supported in partby a grant from the Ministry of Education, Sciences, and Culture ofJapan (#58480104) and a Research Grant for Cardiovascular Diseases(59-C8) from the Ministry of Health and Welfare of Japan.
Reprint requests to Masashi Imai, M.D., Department of Pharmacol-ogy, National Cardiovascular Center Research Institute, 5-7-iFujishirodai, Suita, Osaka 565, Japan.
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