The cortical thick ascending limb and early distal convoluted tubule in the urinary concentrating mechanism

Kidney International, Vol. 31 (1987), pp. 590—596

The cortical thick ascending limb and early distal convoluted tubule in the urinary concentrating mechanism RAINER GREGER and HEIN0 VELAZQUEZ Max Planck Inst itut für Biophysik, Kennedyallee 70, D-6000 Frankfurt 70, Federal Republic of Germany, and Yale University School of Medicine, Department of Internal Medicine, 333 Cedar St., New Haven, Connecticut, USA

This chapter reviews the process of dilution of tubule fluid by the cortical thick ascending limb of the ioop of Henle and by the early distal convoluted tubule. After a description of the cellular

nantly, if not exclusively, through the cells and requires a

mechanisms involved, the factors (pentubular as well as luminal) that modulate the rate of NaCl reabsorption are discussed. Next, the interplay of the cortical thick ascending limb and early distal tubule and their contribution to the urinary concentrating mechanism will be summarized. Complex aspects, such as the function of the two nephron segments in situ and under a variety of conditions, are beyond the scope of this article and will be covered in part by other contributors to this

uptake in the thick ascending limb was originally described for the Ehrlich ascites tumor cell [7], and shortly thereafter for the cortical thick ascending limb [8], and was then confirmed for the medullary thick ascending limb [2] and many other epithelia (which have been referred to, collectively, as "chloride transporting epithelia" [1]). The mechanism of NaCl uptake in the early distal tubule, however, is different. Recent experiments in the rat tubule (in situ and in vivo) suggest that this process is not bumetanide sensitive [9] (vide infra). Furosernide does reduce NaCl transport by a small amount, but at a relatively high dose (0.1 mmolfliter) which may block other cotransport systems. In in vitro perfusion of the rabbit early distal convoluted tubule, we have shown [10] that the luminal membrane has a very high

symposium.

The cellular mechanisms of tubule fluid dilution in the cortical thick ascending limb and in the early distal tubule

Figure 1 depicts cell models for the mechanism of NaCl

carrier—mediated uptake mechanism across the luminal mem-

brane. The Na2ClK carrier that is responsible for the

reabsorption in the two nephron segments, Whereas the model electrical resistance, one to two orders of magnitude higher for the cortical thick ascending limb is well supported by recent than that of the basolateral membrane. The transepithelial experimental work [1], the model for the early distal tubule is voltage is low (——SmV) and the transepithelial electrical resisttentative and preliminary. The model for the cortical thick ance is high (>100 ohm cm2). In view of these results, and since ascending limb is essentially identical to that suggested for the the transepithelial transport rate of NaCL is comparable to that medullary thick ascending limb [2, 3]. However, it should be of the cortical thick ascending limb [10], a strictly electroneutral noted that different species may express some divergence. For mechanism of cellular NaCI uptake has to be postulated. example, the rabbit cortical thick ascending limb appears to Further, this mechanism cannot be the Na2ClK carrier reabsorb NaC1 exclusively via the Na2ClK cotransporter since it is not sensitive to bumetanide and related compounds. [1, 4]. In the mouse this apparently is not the case since up to It is not clear to date whether the NaC1 uptake is via simple 50% of the NaC1 is reabsorbed via the double exchanger NaCl carrier, as has been suggested for the urinary bladder of

system: Na/H, C1/HC03. The rates of these two exchang- the flounder [11], or whether it represents the synchronous

ers seem to be similar so that tubule fluid is neither acidified nor operation of the double exchanger system Na/H and alkalinized [5]. In the rat, as in the mouse, a double exchanger Cl/HCO3-. In the thick ascending limb a large portion of the luminal system is present, but the two components operate at different chloride uptake is balanced by conductive chloride exit across rates such that the tubule fluid is acidified [6]. In view of these species differences, the model for the cortical thick ascending the basolateral membrane. This pathway can be blocked by

limb depicted in Figure 1 has to be regarded as a simplified chloride channel blockers. In the early distal tubule this chloride pathway is absent. Instead, the main conductance of the scheme. The model for the early distal tubule has much in common basolateral membrane is to potassium [12]. Chloride channel with that for the thick ascending limb. In both cases, the blockers have no effect on the basolateral membrane voltage mechanisms of luminal NaCl uptake are energized by the action (unpublished data). The existence of a KC1 symport in the of the (Na + K)-pump located in the basolateral membrane. basolateral membrane of the cortical thick ascending limb as a Secondly, the transport of chloride seems to occur predomi- means of potassium recirculation and chloride exit is supported by the fact that the potassium conductance of this membrane is not sufficiently large enough to allow for the recirculation of the Received for publication July 9, 1986

potassium taken up by the (Nat +K)-pump [1]. In the early

© 1987 by the International Society of Nephrology

distal tubule this KC1 symport in the basolateral membrane is

590

591

Urinary concentrating mechanism Cortical thick ascending limb

Early distal convoluted tubule

Na Fig. 1. Cellular models for the mechanisms of sodium chloride reabsorption in the cortical thick ascending limb of Henle's loop and in the early distal convoluted tubule. Symbols are: (Na+K)-pump; ( (3') carrier systems; (—4-. diffusive pathways. The lower part of the figure gives estimates of the voltages across the cell membranes. With symmetric Ringer—type solutions, the transepithelial voltages in the cortical thick ascending limb and in the early distal convoluted tubule are +5 to +10 mV, and —5 mV respectively. Note that the paracellular shunt pathway is sodium conductive in the cortical thick ascending limb and poorly permeable in the early distal convoluted tubule.

Na *

2 CL

K

postulated to exist since potassium recirculation cannot occur across this cell membrane via the potassium conductance. At first, this may appear paradoxical. However, recall that the voltage across this cell membrane is —85 to —90 mV, and that the electrochemical driving force for potassium exit via the conductance is probably only a few mY. Furthermore, such a KC1 symport could account for the chloride exit, since no other significant conductance but that to potassium was demonstrable in the basolateral cell membrane [10, 12]. Luminal factors determining the rate of NaCI reabsorption in the cortical thick ascending limb and in the early distal tubule The main determinant of NaCI reabsorption in both nephron

250

Na *

K*

/

/

— —— —CL

200 — —

" 150 E

/

// / / //

100

50

/ /,

/

segments is the tubular load of NaC1. The contribution to luminal dilution by both nephron segments is determined by the

NaC1 load delivered from the respective upstream segment. Obviously, the uptake of NaCl across the luminal cell membrane is a saturable process [1] (Fig. 2). Below a certain luminal

NaCl concentration little will be reabsorbed, and above this

0• 0

100

50

150

mmol/liter

Fig. 2. The dependence of the equivalent short circuit current (transepithelial voltage divided by transepithelial electrical resistance) = I,,,

concentration the reabsorption increases with the load. At even on luminal concentrations of K, Na, and C1 in the cortical thick higher concentrations a maximal reabsorption is achieved. The ascending limb of Henle's loop of the rabbit. The data are taken from load to the cortical thick ascending limb is increased whenever [1, 28, 42]. Note that the Isc corresponding to the active reabsorption of the medullary portion of the thick ascending limb is over- sodium chloride [1j will only be limited by the luminal chloride concentration, since the apparent affinities to Na and K are ex-

whelmed by an increased load from an upstream segment. It

could also be that the reabsorption in the medullary thick tremely high. ascending limb is reduced by some other factor, such as in the rodent medullary thick ascending limb by a reduction in the level of antidiuretic hormone [2, 3, 13], or by the so called loop diuretics. In the latter case, however, the cortical thick ascending limb will be unable to cope with the increased NaC1 load, because the loop diuretics will also block NaC1 reabsorption there. Consequently, a large NaCl load will be delivered to the early distal tubule. Because the NaC1 uptake mechanism in the distal tubule is not very sensitive to loop diuretics, an increased distal NaC1 reabsorption will result, which will blunt the di-

The low osmolality of the fluid appearing in the early distal tubule was first documented by Wirz some 30 years ago [14]. This was one of the key findings that led to the concept of the urinary concentrating mechanism. The measured NaC1 concentrations in early distal fluid range between 20 and 50 mmollliter

under normal in vivo conditions [15]. In in vitro perfusion

experiments in the cortical thick ascending limb, however, far higher values have been found [16]. We have repeated these uretic effect and will also lead to an increased secretion of experiments and have reduced the rate of luminal perfusion to potassium in the distal nephron. very low levels. Obviously a collection of perfusate was impos-

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Greger and Velázquez

Peritubular factors regulating the rate of NaCl reabsorption in transepithelial lumen positive voltage of approximately the cortical thick ascending limb and in the early distal tubule +3OmV, and from the known permselectivity of the paracellular The peritubular factors include hormones, specific ions such shunt pathway [1], it can be calculated that this voltage reflects as calcium, magnesium, potassium, and protons, osmolality, a luminal NaC1 concentration of 20 to 30 mmollliter. This specific agents such as chloride channel blockers, and metavoltage is a steady state value that would correspond to the zero bolic substrates. net flux of NaCl. Thus, at this low NaC1 concentration, luminal Na2ClK uptake will be reduced to some 20 to 25% of Hormones

sible in these experiments [17]. However, from the maximal

control (Fig. 2). The absorptive flux will be balanced by a backflux of equal magnitude. The backflux most likely occurs through the paracellular shunt pathway. For sodium the transepithelial driving force favoring backflux is relatively small, since the chemical gradient is partially balanced by the lumen positive voltage, The partial conductance to sodium is high [1]. For chloride the situation is opposite: the driving force is high,

Unlike the medullary thick ascending limb of rodents [18], the medullary thick ascending limb of the rabbit and of man and the cortical thick ascending limb of all species investigated thus far

have only a limited number (rabbit) or do not have (man) receptors for antidiuretic hormone [131. However, receptors for other hormones are present in the cortical thick ascending limb:

and the conductance small. This pump/leak condition at a glucagon, catecholamines, calcitonin, parathyroid hormone (PTH) [13]. The effects of these hormones in vivo will be reduced uptake rate is energy saving, since the cellular uptake

discussed elsewhere in this symposium. Suffice it to state that

of sodium and thus the rate of (Na +K)-pump activity is PTH or calcitonin or the second messanger cAMP in the form of reduced to about one fourth. It should be emphasized that the

the membrane permeable dibutyryl-cAMP had no effect in the

reduction of the uptake rate via the Na2ClK carrier is rabbit on transepithelial voltage, transepithelial electrical resist-

caused mainly by its chloride concentration dependence. The ance, the rate of NaC1 reabsorption, or the permselectivity to uptake rate is half maximal at some 40 to 50 mmol/liter chloride NaCl of the paracellular shunt pathway (18, 20, and unpub(Fig. 2). The luminal sodium and potassium concentrations are lished observations in the authors laboratory). However, since not limiting since the apparent KM values for these ions are on the sensitivity of the methods used to measure NaC1 absorption the order of 1 to 3 mmol/liter (Fig. 2). may not have been adequate to detect small effects, these data The interplay between the medullary and cortical thick should be interpreted with caution. The hormonal effects in the ascending limb has been interpreted as a combination of a high early distal tubule have thus far not been studied by in vitro capacity, low affinity (medullary) and a low capacity, high perfusion. Thus far, no direct evidence exists that steriod hormones act affinity (cortical) system [18]. This is substantiated by the fact that the net rates of NaC1 reabsorption are higher in the on the cortical thick ascending limb of the rabbit. However, this medullary than in the cortical portion of the thick ascending may be a problem of the species used, since mineralocorticoid limb, and that the density of mitochondria and the concentra- binding is demonstrable in the cortical thick ascending limb of tion of (Na + K)-ATPase per tubule length is higher in the rats [21], and since mineralocorticoid effects are clearly demonmeduilary segment. However, the differences may not be as strable in the segment analogous to the thick ascending limb in the amphibian nephron, the diluting segment. Mineralocortimarked as they have been stated previously [1]. In the early distal tubule we have little knowledge of the coids increase the luminal uptake of sodium via a Na/H concentration dependence of NaCl reabsorption. It has been exchanger. In addition, they stimulate secretion of potassium, suggested that chloride leaks back into the lumen in this since increased proton extrusion alkalinizes the cell and augments the luminal potassium conductance [22]. Mineralocornephron segment if a maximal gradient for NaC1 has been built ticoid effects in the early distal convoluted tubule are still up by the cortical thick ascending limb. Thus, the maximal controversial. Mineralocorticoid receptors are demonstrable NaC1 gradient sustained by the early distal tubule may be lower [21], but the effects of mineralocorticoids on transport or than that generated by the cortical thick ascending limb [15]. voltage in this portion of the distal tubule have not been Other luminal factors that influence the rate of NaC1 reab- documented unequivocally [23]. Since only a portion of this sorption in the cortical thick ascending limb are the tubule fluid nephron segment is accessible to free flow micropuncture, and concentrations of calcium and magnesium and (probably) pH. since that portion is very short, in vitro perfusion experiments A reduction of luminal fluid calcium concentration below 2 on this nephron segment need to be performed to examine the mmollliter increases the sodium conductance of the paracellular effects of various hormones. shunt pathway and vice versa [1]. Magnesium has an identical Peritubular calcium and magnesium effect. This observation is similar to that made in the proximal nephron [19], and it appears to involve the titration of negative In the cortical thick ascending limb an increase in the calcium fixed charges in this pathway. As in the proximal tubule, or magnesium concentration (above 2 mmollliter) in the perituacidification of luminal fluid might have a similar effect. A bular fluid reduces the shunt conductance to sodium. This effect reduction of the paracellular sodium conductance compromises is identical to that described above for the luminal fluid. In the NaCl reabsorption inasmuch as the passive component of early distal tubule, removal of magnesium from the blood side sodium reabsorption is reduced. Thus, the amount of NaC1 has remarkable effects which are not yet understood (Table 1). reabsorbed per cycle of the (Nat +K)-pump, which is 6:1 The transepithelial voltage increases and the transepithelial maximally (mol NaCI per mol ATP) under control conditions resistance falls slightly as does the basolateral membrane voltage. This occurs when the magnesium concentration is reduced [1], may be reduced to as little as 3:1.

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Urinary concentrating mechanism

Table I. Effect of basolateral magnesium on rabbit early distal tubule perfused in vitro Solution

I, +Mg II, —Mg

R,

Vte

Ohm cm2

mV —6.5 —9.7

n=

0.96

iiia

39

105.2

99.9

n=

VbI

mV

9.91

—86.2

9.52

—83.1

28

n=

1.15 1.69a

49

Solutions I and II are artificial solutions that resemble an ultrafiltrate of plasma. +Mg and —Mg indicate presence or absence of 1 mM Mg.

Abbreviations are: Vte, transepithelial voltage; Rta, transepithelial resistance; YbI, basolateral membrane voltage, a Indicates a significant difference, P <0.05, II vs. I, t-test for paired data

R2

x no no no CH2 C2H4 C3H6 C4H8

R1

H H NO2 NO2 NO2 NO2 NO2

R2 H CI H H H H H

# 11

208

IC

2 4109.1O6 410

71

31O 31O

76 129 130 145

8.1O8

1.810

3. Chloride channel blockers in the cortical thick ascending limb of below 1 mmollliter. Removal of magnesium on the luminal side Fig. Henle's loop of the rabbit. The upper part gives the schematic struc-

has no effect. It is still unclear how changes in peritubular tural formula. X is an aliphatic chain, RI and R2 are substitutions, # is

magnesium cause these events. We also cannot yet determine the identifying number of the substance, IC50 is the peritubular concenwhether this marked increase in transepithelial current flow tration necessary to block 50% of the equivalent short circuit current

induced by a reduction in peritubular magnesium has any (legend to Fig. 2). The data are taken from [30 and 31]. physiological relevance. Decreasing the peritubular pH below approximately 6.8 leads

to a reduction of NaCl reabsorption in the cortical thick conductance of the amphibian diluting segment and also has a ascending limb (author's unpublished observation). An increase in pH above the normal value, at least in the amphiuma diluting segment, has been claimed to increase the luminal membrane conductance to potassium [24]. This will result in an increased secretion of potassium. Peritubular potassium has a very complex effect on the rate of NaCl reabsorption in the thick ascending limb. An increase in potassium concentration reduces the reabsorption of NaCl [25]. It was suggested that this occurs because the increased potas-

sium concentration impedes the electroneutral KC1 exit step for chloride (Fig. 1). To elicit this effect, a rather high peritubular concentration of potassium is required. Also it has been reported that a reduction in pentubular potassium concentration compromises the diluting ability of the thick ascending limb [26]. These experiments have been performed in the intact rat, where a marked effect occurs at a plasma potassium concentration of 2 to 3 mmollliter. We have performed in vitro perfusion experiments, both in the medullary and in the cortical thick ascending limb, and found that NaC1 reabsorption, as measured by the equivalent short circuit current, remains unaltered when bath potassium concentrations are decreased to as low as 1.5 to 2 mmollliter [I]. Only at potassium concentrations lower than 1.5 mmollliter did the rate of active transport fall. It is possible

limited effect in the thick ascending limb. We have searched for more potent compounds [31—33] and have found strongly acting

blockers of the phenylamine-2-benzoate type (Fig. 3). These substances interact reversibly with the chloride channel. By blocking this channel, the basolateral membrane resistance and,

to a moderate degree, the transepithelial electrical resistance, increase. The transepithelial voltage collapses and the cell hyperpolarizes. The effect on net reabsorption of NaCl has not yet been determined, but it is to be predicted that the fall in the active transport potential is paralleled by a fall in net reabsorption of NaCI. Currently, it is unclear whether these compounds or derivatives thereof will become pharmacologically important. Substrate uptake

For the cortical thick ascending limb to absorb NaC1 normally, substrates such as pyruvate, acetate, and D-glucose must be supplied continuously to the pentubular fluid [34]. The addition of substrates to the lumen is ineffective. The collapse

of active transport upon substrate removal is rapid (Fig. 4): Within three minutes of substrate free perfusion, active transport is reduced to approximately 50%, and the cells start to swell and depolarize [35]. This finding may explain why this that the latter effect is caused by an inhibition of the (Na segment is so vulnerable to ischemia. The implication is that +K)-pump. The results obtained in the intact animal are ischemia of the renal medulla, such as in acute renal failure, will therefore most likely due to an indirect effect of hypokalemia. rapidly compromise the urinary concentrating mechanism [36]. Peritubular osmolality Peritubular hyperosmolality reduces the rate of NaCl reabsorption by the medullary thick ascending limb [27]. This effect is also present in the cortical thick ascending limb, but is less marked [281.

Chloride channel blockers A new class of substances has emerged recently from initial studies with anthracene-9-carboxylate [29, 30]. Anthracene-9carboxylate has some blocking effect on the basolateral chloride

Diuretic effects in the cortical thick ascending limb and in the early distal tubule Furosemide and related compounds [37, 38] interfere with the

chloride binding site of the Na2ClK cotransporter. Thus, the sodium entry into thick ascending limb cells is blocked. Several aspects of the interaction of these substances with the thick ascending limb cell have been examined in detail recently.

It has been proposed that the inhibition of sodium entry by these diuretics also reduces dramatically the oxygen consump-

594

Greger and Velázquez

so

No substrate

250-

+

IC50

9

7.1.10_B

H

SO2NH2

200-

so2

, 150-

a coo

100 -

N4S022 50

BUM

210

6

0—2

0

2

4

6

8

10

Time, mm

4. Dependence of the equivalent short circuit current Isc (Fig. 2 legend) of cortical thick ascending limbs of Henle's loop of the rabbit on the presence of metabolic substrates. The shaded area corresponds to mean values SEM. At zero time all metabolic substrates were Fig.

•cr

H N

removed on the peritubular side. Data taken from [34]. Note that removal of peritubular substrates leads to a precipitous fall in the

SO2NCONHC3H7 TOR

coo

equivalent short circuit current, corresponding to a parallel fall in the rate of active sodium chloride reabsorption.

CN tion and the (Nat +K)-pump activity [4, 39, 40]. These observations are of practical importance since they imply that ischemic damage to the thick ascending limb can be reduced or

even prevented if a diuretic of this type is applied prior to

SO2NH2

PIR

6

ischemia of the renal medulla [35]. Fig. 5. Inhibitors of the Na2ClK cotransporter in the cortical thick The structure—function relationship for this group of sub- ascending limb of Henle's loop of rabbit nephron. The first column

stances has been determined [37]. Some of the most potent derivatives are summarized in Figure 5. At concentrations below 10 molIliter substances interact specifically with the

Na2ClK cotransporter. At much higher concentrations other transport proteins can also be inhibited. Since these

gives the structural formula. The IC0 values correspond to the concentration in the luminal perfusate necessary to reduce the equivalent short circuit current (Fig. 2 legend) to 50% of control. 9, BUM = bumetanide, and PIR = piretande are taken from [37], TOR = torasemide are unpublished data from the author's laboratory, Note that the blockers

of the Na2ClK cotransporter share structural similarities with the chloride channel blockers (Fig. 3).

diuretics can block virtually all NaCl reabsorption in both the medullary and the cortical thick ascending limbs, they clearly can compromise the counter—current multiplication system. These substances remain the most potent diuretics. cations [1]. This component, however, appears to be less It may be useful to consider the mechanism by which these important than the transcellular reabsorption of potassium

diuretics cause a profound kaliuresis in the loop of Henle. which occurs via the Na2ClK cotransporter. The diffusion Normally, the fluid emerging from the cortical thick ascending

voltage across the shunt pathway leads to a hyperpolarization

limb is hypotonic, and the low NaC1 concentration is also of the luminal membrane. Whereas the backflux of potassium responsible for the marked increase in the lumen positive from cell to lumen balances the rate of uptake via the voltage in the cortical thick ascending limb. The reason for the Na2ClK cotransporter when symmetric NaCl solutions are large lumen—positive voltage of up to 30 mY is a sodium present, a hyperpolarization of the lumen membrane voltage diffusion potential generated by the sodium—selective paracellular shunt pathway [1]. This high transepithelial voltage leads

to substantial potassium reabsorption. The mechanisms involved can be deduced by inspecting Figure 1. The increased transepithelial voltage drives potassium to the blood side through the paracellular pathway, which is permeable to small

(such as that present when the lumen fluid is dilute) by only 5 mV could reduce the backflux of potassium by approximately 50%. The rate of potassium reabsorption in the thick ascending limb depends, therefore, on the axial transepithelial—voltage profile. If the reabsorptive capacity of the medullary and the cortical thick ascending limb is not saturated, passive potas-

595

Urinary concentrating mechanism

sium reabsorption will be high and it will occur mostly in the cortical thick ascending limb. If the NaC1 reabsorptive capacity is saturated, little potassium reabsorption will occur in the thick

ascending limb. The diuretics which interfere with the Na2ClK cotransporter will abolish NaCL reabsorption in the thick ascending limb, and therefore the reabsorption of potassium normally present. The increased NaC1 and volume load delivered to the distal nephron, will increase distal tubular potassium secretion. The loop diuretics do not act in the early distal tubule. Nor are amioride and related compounds effective in this portion of the distal nephron [9, 41]. On the other hand, the thiazides, which act from the lumen side of distal cells and block the reabsorption of NaC1, are ineffective in the thick ascending limb [9,37]. The cellular mechanism of the action of thiazides is still

References 1. GREGER R: Ion transport mechanisms in thick ascending limb of Henle's loop of mammalian nephron. Physiol Rev 65:760—797, 1985 2. HEBERT SC, ANDREOLI TE: The effects of antidiuretic hormone on

cellular conductive pathways in mouse medullary thick ascending limbs of Henle. II. Determinants of the ADH-mediated increases in

transepithelial voltage and in net Cl absorption. J Membr Biol 80:221—233, 1984

3. SCHLATTER E, GREGER R: cAMP increases the basolateral C1-

conductance in the isolated perfused medullary thick ascending limb of Henle's loop of the mouse. PflOgers Arch 405:367—376, 1985 4. GREGER R, SCHLATFER E: Cellular mechanism of the action of loop

diuretics on the thick ascending limb of Henle's loop. Klin Wochenschr 61:1019—1027, 1983 5. FRIEDMAN PA, ANDREOLI TE: C02-stimulated NaCl absorption in

the mouse renal cortical thick ascending limb of Henle. J Gen Physiol 80:683—711, 1982

unclear. They most likely interfere with a sodium chloride

6. GooD DW, KNEPPER MA, BURG MB: Ammonia and bicarbonate

uptake mechanism. It has been reported recently that thiazides block an electroneutral NaCI cotransport system present in the apical membrane of the flounder urinary bladder [11].

7. GECK P, PIETRZYK C, BURCKHARDT B, HEINZ E: Electrically silent

Regulation of the function of the cortical thick ascending limb and of the early distal tubule in the counter current system

The cortical thick ascending limb can be termed a buffering system. Whenever the medullary thick ascending limb is over-

whelmed by a large NaC1 load (caused by impaired NaC1 reabsorption in an upstream segment or in the medullary thick ascending limb) the cortical portion will try to cope with this additional load. This necessarily will be paralleled by a reduction of the transepithelial NaCl gradient, and, consequently, will reduce the diluting capacity. The short early-distal tubule serves a similar function but its role is not as important. Antidiuretic hormone, which has a pronounced effect on the

medullary portion of the thick ascending limb, seems to be without effect in the cortical thick ascending limb of the rabbit and the mouse [1, 5]. The effects of other hormones such as catecholamines, glucagon, calcitonin, parathyroid hormone,

and steroid hormones on NaC1 absorption have not been

studied in great detail in in vitro perfused cortical thick ascending limb segments. Several pentubular factors are of primary physiological relevance to the function of the cortical thick ascending limb. The peritubular circulation must remain uninterrupted and deliver substrates continuously. The osmolality of the peritubular fluid must be maintained. Finally the concentrations of calcium, magnesium, potassium and protons contribute importantly to the regulation of cortical thick ascending limb function. Factors regulating the function of the early distal tubule are less clear. The functional and morphological heterogeneity of the distal nephron makes necessary a reexamination of all the different portions utilizing microdissected and clearly—defined nephron segments.

transport by thick ascending limb of rat kidney. Am J Physiol 247(Renal Fluid Electrol Physiol 16):F35—F44, 1984

cotransport of Na, K and Cl- in Ehrlich ascites cells. Biochim Biophys Acta 600:432—447, 1980 8. GREGER R, SCHLATTER E: Presence of luminal K, a prerequisite for

active NaC1 transport in the cortical thick ascending limb of Henle's loop of rabbit kidney. Pflugers Arch 392:92—94, 1981 9. VELAZQUEZ H, WRIGHT FS: Effect of diuretic drugs on Na, Cl, and K transport by rat renal distal tubule. Am J Physiol 250 (Renal Fluid Electrol Physiol 19) Fl013—Fl023, 1986 10. VELAZQUEZ H, GREGER R: Electrical properties of the early distal convoluted tubules of the rabbit kidney (abstract). Renal Physiol (in press)

11. STOKES JB: Thiazides inhibit neutral NaCl cotransport in the flounder urinary bladder. (abstract) Proc 9th mt Congr Nephrol, Los Angeles, 1984, 434A 12. VELAZQUEZ H, GREGER R: Influences on basolateral K-conductance of cells of early distal convoluted tubule (abstract). Kidney In: 29:409, 1986 13. MOREL F, IMBERT—TEB0UL M, CHABARDES D: Distribution of

hormone—dependent adenylate cyclase in the nephron and its physiological significance. Ann Rev Physiol 43:569—581, 1981

14. Wntz H: Der osmotische Druck in den kortikalen Tubuli der Rattennieren. Helv Physiol Pharmacol Acta 14:353—362, 1956 15. SCHNERMANN J, BRIGGS J, SCHUBERT G: In situ studies of the distal convoluted tubule in the rat. I. Evidence for NaC1 secretion. Am J Physiol 243 (Renal Fluid Electrolyte Physiol 12):Fl60—Fl66, 1982

16. BURG MB, GREEN N: Function of the thick ascending limb of Henle's loop. Am J Physiol 224:659—668, 1973 17. GREGER R, SCHLATFER E: Mechanism of chloride transport in vertebrate renal tubule, in Chloride Transport Coupling in Biological Membranes and Epithelia, edited by GERENCSER GA. Amsterdam, Elsevier, 1984, p. 271—346

18. BURG MB: Thick ascending limb of Henle's loop. Kidney mt 22:454—464, 1982

19. FROMTER E, MULLER CW, KNAUF H: Fixe negative Wandladungen im proximalen Konvolut der Rattenniere und ihre

Beeinflussung durch Calciumionen, in Aktuelle Probleme des

Elektrolyt- und Wasserhaushalts, Nierenbiopsie, edited by WATSCHUNGER B. Vienna, Wiener Med Akad, 1969, pp. 61—64

20. BOURDEAU JE, BURG MB: Effect of PTH on calcium transport

across the cortical thick ascending limb of Henle's loop. Am J Physiol 239 (Renal Fluid Electrolyte Physiol 8): Fl2l—l26, 1980

Acknowledgment This study was supported by Deutsche Forschungsgemeinschaft

21. FARMAN N, BONVALET JP: Aldosterone binding in isolated tubules

III: autoradiography along the rat nephron. Am J Physiol 245 (Renal Fluid Electrolyte Physiol 14):F606—F6l4, 1983

Gr480/6-4.

22. OBERLEITHNER H, LANG F, MES5NER G, WANG W: Mechanism of

Reprint requests to Heino Velázquez, Ph.D., Yale University School of Medicine, Department of Internal Medicine, 333 Cedar Street, New Haven, Connecticut 06510, USA

23. KAISSLING B: Structural aspects of adaptive changes in renal electrolyte excretion. Am J Physiol 243 (Renal Fluid Electrol

hydrogen ion transport in the diluting segment of frog kidney. Pflugers Arch 402:272—280, 1984 Physiol l2):F21 1—F226, 1982

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Greger and Velazquez

24. GUGGINO WB, STANTON BA, GIEBISCH G: Regulation of apical

potassium conductance in the isolated early distal tubule of the Amphiuma kidney. (abstract) Biophys J 37:338, 1982 25, STOKES JB III: Consequences of potassium recycling in the renal medulla. Effects on ion transport by the medullary thick ascending limb of Henle's loop. J C/in Invest 70:219—229, 1982 26. GUTSCHE HU, PETERSON LN, LEVINE DZ: In vivo evidence of impaired solute transport by the thick ascending limb in potassiumdepleted rats. J C/in Invest 73:908—916, 1984 27. HEBERT SC, CULPEPPER RM, ANDREOLI TE: NaCI transport in mouse medullary thick ascending limbs. III. Modulation of the ADH-effect by peritubular osmolality. Am J Physiol 241 (Renal Fluid Electrolyte Physiol lO):F443—F45 1, 1981 28. GREGER R, SCHLATTER E, LANG F: Evidence for electroneutral

sodium chloride cotransport in the cortical thick ascending limb of Henle's loop of rabbit kidney. Pflugers Arch 396:308-3 14, 1983

29. OBERLEITIINER H, RITTER M, LANG F, GUGGINO W: Antracene-

9-carboxylic acid inhibits renal chloride reabsorption. Pflugers Arch 398:172—174, 1983

30. GREGER R: The Na2ClK carrier of the lumen membrane of the diluting segment of rabbit kidney. Fed Proc 43:2473—2487, 1984 31. WITTNER M, LANG Hi, ENGLERT HC, DI STEFANO A, SCHLATTER

E, GREGER R: Cl--channel blockers. Structure activity relationships of diphenylamine-2-carboxylate (DP-2-C) derivatives. (abstract) Pflugers Arch 403:Rl2, 1985 32. WANGEMANN P, WITTNER M, DI STEFANO A, ENGLERT HC, LANG Hi, SCHLATTER E, GREGER R: Cl channel blockers in the

thick ascending limb of the loop of Henle. Structure activity relationship. Pflugers Arch (in press) 33. D STEFANO, WITTNER M, SCHLATTER E, LANG Hi, ENGLERT H,

GREGER R: Diphenylamine-2-carboxylate, a blocker of the C1conductive pathway in C1-transporting epithelia. Pflugers Arch

405(Suppl 1):S95—SlOO, 1985 34. WITTNER M, WEIDTKE C, SCHLATTER E, DI STEFAN0 A, GREGER

R: Substrate utilization in the isolated perfused cortical thick ascending limb of rabbit nephron. Pflügers Arch 402:52—62, 1984 35. GREGER R, SCHLATFER E, WITTNER M: Cellular mechanism of furosemide-like diuretics in the thick ascending limb of the loop of Henle, in Diuretics: Chemistry, Pharmacology, and Clinical Applications, edited by PUSCHETT iB, GREENBERG A. Amsterdam, Elsevier, 1984, pp. 215—221 36. BREzI5 M, ROSEN S, SILVA 5, EPSTEIN F: Renal ischemia: A new perspective. Kidney mt 26:375—383, 1984 37. SCHLATTER E, GREGER R, WEIDTKE C: Effect of "high ceiling" diuretics on active salt transport in the cortical thick ascending limb of Henle' s loop of rabbit nephron. Correlation of chemical structure and inhibitory potency. Pflugers Arch 396:210—217, 1983 38. HANNAFIN J, KINNE—SAFFRAN E, FRIEDMANN D, KINNE R: Presence of a sodium—potassium--chloride cotransport system in

the rectal gland of SQUALUS ACANTHIAS. J Membr Biol 73:73—83, 1983

39. EVELOFF J, BAYERDORFFER E, SILVA P, KINNE R: Sodium—

chloride transport in the thick ascending limb of Henle's loop. Oxygen consumption studies in isolated cells. Pflügers Arch 389:263—270, 1981

40. CHAMBERLIN ME, LEFURGEY A, MANDEL Li: Suspensions of medullary thick ascending limb tubules from rabbit kidney. Am J Physiol 247 (Renal Fluid Electrolyte Physiol 16):F955—F964, 1984 41. COSTANZO LS: Comparison of calcium and sodium transport in early and late distal tubules: Effect of amiloride. Am J Physiol 246: F937—F945, 1985

42. GREGER R: Chloride reabsorption in the rabbit cortical thick ascending limb of the loop of Henle. A sodium dependent process. Pflugers Arch 390:38—43, 1981