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Kidney International, Vol. 32 (1987), pp. 136—150

ROBERT F. PITTS MEMORIAL LECTURE

H secretion in renal cortical tubules: Kinetic aspects1

GERHARD MALNIC

Department of Physiology and Biophysics, Institute Ciencias Biomédicas, Univiersidade de Sao Paulo, Sao Paulo, Brasil

The Robert F. Pitts Memorial Lectureships were founded in 1978, when the many friends and admirers of Robert Pitts
established a fund in memory of that distinguished physiologist. The lecturer is selected by the Renal Commission of the
International Union of Physiological Sciences, with the Chair of Physiology at Cornell University Medical College acting ex
officio. The lecture is presented at each International Congress of Physiological Sciences. At the XXXth Congress held in
Vancouver, Canada, the Pitts Lecturer was Professor Gerhard Malnic from Sao Paulo, Brazil. He delivered the lecture on July
15, 1986.
There are several aspects about the choice of Professor Malnic that seem singularly fitting. First and foremost, he is an
outstanding physiologist who, like Robert Pitts, has contributed much to our understanding of renal function. Of Pitts' many
contributions to physiology, the major one was probably his brilliant elucidation of how the kidney maintains hydrogen ion
balance, and it is in this area that Gerhard Malnic has done most of his original work. Professor Malnic received his major
postdoctoral training in Dr. Pills' department at Cornell. There, under the tutelage of Gerhard Giebisch, Professor Malnic became
first author on a series of three classic papers, published in 1964 and 1966, which conclusively showed, by micropuncture
techniques, how the kidney handles potassium. And it was again at Cornell, in a later sojourn as Visiting Scientist, that Professor
Malnic began his long and fruitful studies on the renal handling of hydrogen ion.
Gerhard Malnic was born in Italy, of Austrian parents. In 1956, he became a citizen of Brazil, where he received his medical
training. In the intervening thirty years, he has become a leader in South American physiology and science. He has served as
Secretary—General of the Latin American Association of Physiological Sciences and as President of the Brazilian Physiological
Society, and he is a member of the Academies of Sciences of both Brazil and Latin America. Currently, he holds the position of
Professor and Chair of the Department of Physiology and Biophysics at the University of Sao Paulo.
Professor Malnic's work on the renal handling of hydrogen ion has progressed from a methodical description of acidification
along the nephron to exploring the detailed mechanisms that govern the tubular secretion of hydrogen ion. It is the latter aspect
that formed the major topic of his Robert F. Pitts Lecture.
HEINZ VALTIN
Chair, Renal Commission
Previous Robert F. Pitts Memorial Lecturers
1980 RoIf Kinne, Federal Republic of Germany
1983 James A. Schafer, U.S.A.

the site of a well defined mechanism of Na/H exchange in the

The kidney plays a key role in the regulation of the acid—base
brush—border cell membrane, a transport mechanism that has
balance of the body. Not only is it responsible for the elimina-
tion of fixed acid and for the recovery of most of the filtered
been studied by micropuncture, microperfusion and membrane
bicarbonate, but also for the formation of ammonia and the
vesicle techniques [5—11]. Na/H exchange has also been found
ultimate elimination of ammonium ions. All these processes
in the thick ascending limb of Henle's loop [12, 13]. A minor
help maintain stable body fluid pH. portion of H ion secretion may be mediated, in the proximal
tubule, by an ATP-driven proton pump in the brush border
In the last years, our knowledge of the mechanisms responsi-
membrane [14]. While it is accepted that the most important
ble for these processes has dramatically expanded. It is now
fraction of HCO3 reabsorption in the proximal tubule is medi-
generally accepted that titratable acid formation, bicarbonate
reabsorption and ammonia excretion largely depend on H ated by H ion secretion, some evidence has been presented,
transport. Active H ion secretion occurs along the whole both in fish [15, 16] and in mammals [17] that a carbonic—anhy-
nephron with the exception of the thin limbs of Henle's loop drase independent mechanism may contribute to bicarbonate
[1—4]. Thus, the proximal convoluted and straight tubules are reabsorption. The nature of this mechanism has not yet been

'This lecture was presented at the XXXth Congress of The Inter-

Received for publication August 26, 1986 national Union of Physiological Sciences, Vancouver, Canada, July 15,
© 1987 by the International Society of Nephrology 1986.

136
 H secretion in renal cortical tubules
3 Na
Na*
nHCO3
A
HCO3
HC03
3 Na
HC03
H like Na and K have been found [26—28]. Such high perme-
Fig. 1. Acidification mechanisms in renal tubule cells. (+) and (—):
rheogenic mechanisms.
fully explained, but due to the finite permeability of proximal
epithelium to bicarbonate, passive movement along a favorable
electrochemical potential gradient is possible and has indeed
been demonstrated in bicarbonate loading or after carbonic
anhydrase inhibition [18]. Transcellular bicarbonate ion trans-
port as such has also been proposed [17, 19, 20], but evidence
for such a process in the proximal tubule is presently lacking.
Evidence does exist for rheogenic ATP-dependent H-ion
secretion along the cortical and medullary collecting duct [21].
This process is in many aspects similar to that described for
turtle bladder, a structure that is the site of a well—developed
acid secretory—mechanism [22]. This preparation has also been
shown to be the site of bicarbonate secretion [23]. More
recently such a mechanism has also been found in the collecting
duct of rabbit and rat kidney in alkalotic conditions [24, 25].
Figure 1 summarizes the more important mechanisms for acid-
ification of tubular fluid in proximal and distal nephron cells.
Besides the basic mechanisms discussed above, properties
that involve the acid/base regulating mechanisms of the neph-
ron such as permeabilities to H and HC03, the base extru-
sion mechanism at the basolateral cell membrane, hormonal
regulation, pathophysiological situations and many others have
also been investigated [2, 4]. In this paper, I will discuss in more
detail some aspects of the cellular mechanisms of renal tubular
acidification that our laboratory has contributed to over the
years, including passive H transfer across proximal epithe-
hum, load dependence of proximal acidification, basolateral
137
membrane base—transfer, and the ability of distal tubules to
reabsorb bicarbonate and to establish pH gradients. All these
mechanisms help us understand how the renal tubule produces
an acid urine and regulates renal acid excretion.
The nature of passive H+ backflux across renal tubule
epithelium
Studies on proximal brush—border membrane vesicles show
that ion translocations by the Na/H exchanger depend on the
prevailing concentration gradients. The resulting ion move-
ments are therefore symmetrical, that is, the system is able to
transfer these ion species bi-directionally, in the direction
determined by the concentration gradient of the coupled spe-
cies. Thus, it is possible to transfer H ions against a gradient by
establishing a Na gradient, or Na by setting up a pH gradient
[6-8].
Since the Na/H exchanger is symmetrical in isolated
brush—border membrane vesicles, it should also be reversible in
the corresponding epithelia, in vivo or in vitro, depending only
on the direction of the existing concentration gradients. Thus,
the exchanger might not only effect H ion secretion, but also,
when H gradients are reversed, contribute to passive H ion
back—flux, that is, to the apparent H ion permeability. Apparent
permeability to H ions has been measured by a number of
workers, and in general values much higher than those for ions
ability may in part be due to a specialized path for these ions
across tubular membranes, which could be the Na/H exchanger
itself. Investigations used different methods to study this aspect
of H ion transport in the in vivo rat kidney, the isolated
perfused rat kidney, isolated perfused proximal tubules of the
rabbit, and in brush—border membrane vesicles.
Schwartz [29, 30] and Hamm et a! [27] studied the
reversibility of the Na/H exchanger by measuring the pH
increase when perfusing in vitro isolated rabbit proximal con-
voluted tubules, subject to transepithelial pH gradients in the
appropriate direction. Schwartz, perfusing these tubules at 38°C
in HCO3-C02-free media, obtained a marked elevation of H
effiux from the lumen (pH 6.7 vs. bath 7.4) when ouabain was
added to the bath, a procedure elevating cell sodium levels. H
effiux decreased by 44% when sodium was substituted by
choline or lithium. H efflux was also reduced when organic
solutes were excluded from the perfusate, but this efflux was
still sensitive to the maneuvers described above. However, a
significant portion of H fluxes remained insensitive to low Na
media. The author concluded that a portion of the H fluxes
along concentration gradients across the luminal cell membrane
flows through the Na/H exchanger. A later study, by Hamm et
a!, however, did not confirm Schwartz's conclusion [27]. These
authors used similar microperfusion procedures, but worked at
both 21 and 37°C, in the latter case adding acetazolamide to
their preparation to eliminate active transport mechanisms.
They obtained W permeability coefficients of 0.15 cmlsec at
21°C, and of 0.31 cm/sec at 37°C. Under both these conditions
they were not able to detect a significant effect of low sodium
media or 10 M amiloride on H ion permeability. The presence
of other buffer systems besides HC03/C02 also affects these
measurements. The undissociated acid form of organic sub-
strates like lactate, citrate and alanine contributes to H ion
138 Malnic

A Secretion Back—flux

100
'P

80

Pert. Block
60
8
B
40
z
20

6
10
0
C
Fig. 3. H ion fluxes in proximal tubule of isolated perfused rat kidney
E during control ( 20 mM phosphate Ringer's) and low—sodium ( Na
5 substituted by choline, iO M amiloride in lumen) conditions. JH in %
of control. Data from [37].
8 1/2 6.69 S
00
I 2
Table 1. H ion permeability in proximaI tubules of mammalian
kidney
z
Co

PH,
cm/sec Reference
0 In vivo rat kidney
Doubly perfused tubules 0.53 37
10 20 30 Isolated perfused rat kidney," 37°C 1.39 37
Time, sec 37°C, low Na, A 0.52 37
20°C, control 0.30 37
Fig. 2. Representation of method to measure renal tubule acidification Isolated perfused rabbit proximal tubule
kinetics. A. schematic representation of experiment: I - double—barrel- 0.48 30
37°C, no organic solutes
led microperfusion pipette; 2 - capillary perfusion micropipette; 3 - pH 37°C, no organic solutes, low Na 0.33 30
microelectrode; A, amplifier. B. record of luminal pH changes during 37°C, no organic solutes 0.31 27
perfusion with alkaline solution. C. plot of log of buffer concentration 0.15 27
21°C, no organic solutes
against time; 112, acidification half—time. Rabbit brush—border, 20°C 0.005 28
Lipid bilayer, pH 7, 24°C 0.0001 38
° Luminal and capillary perfusion with 20 m phosphate Ringer's
transfer. The contribution of phosphate movement across the
b Luminal perfusion with 20 m phosphate Ringer's, 5.5 mat glucose
epithelium was insignificant compared to the observed rates of added. Low Na: 0.25 mat lumen, 10.7 mM vascular, iO M amiloride
H ion flux [27, 31, 32]. added to luminal solution. PH = JH' [Hi, cm/sec
In our laboratory we applied a stopped—flow microperfusion
method to the analysis of H ion fluxes both of the in vivo and
isolated—perfused rat kidney [33, 34]. The kinetics of acidifica- In order to reduce the presence of organic buffer systems and
tion or alkalinization of luminally—injected fluid columns allow to be able to work in a low HC03/Cfl2 system we used the
us to evaluate H ion fluxes as well as the stationary pH isolated perfused rat kidney preparation as described by Maack
gradients, and H ion efflux from the tubular lumen. Figure 2 [36]. This preparation allows us to work at different tempera-
gives a composite view of this method. By injecting a solution tures, which is especially useful for the questions we are
more acid than the luminal steady—state level (pH 5.5 to 6.2) and presently addressing.
blocking this solution within the lumen, we can measure the Figure 3 shows the role of a low sodium medium and of
rate of alkalinization of this fluid. In in vivo experiments with addition of M amiloride on both H ion secretion and
blood—perfused capillaries, rates of alkalinization are initially back—flux. H ion secretion is entirely abolished in this prepara-
very high, corresponding to an apparent H-ion permeability of tion, and H ion back—flux is reduced to 38% of the control level.
several cm/sec [261. In this measurement, however, bicarbonate Table 1 gives H-ion permeability values obtained in the in
back—flux from capillary to lumen is included, a factor acceler- vivo doubly—perfused proximal tubule, in the isolated—perfused
ating the observed alkalinization [35]. The presence of other rat kidney, in isolated—perfused rabbit proximal—tubule, in
buffer systems originating from blood may also increase proximal brush border and in lipid bilayer membranes. The
alkalinization rates by contributing permeant nonionic acids to Table shows that, in comparable conditions, the W permeabil-
the medium. ity of proximal tubular epithelium is somewhat more elevated in
 H secretion in renal cortical tubules 139

>
1.0

90V E RH

>
0
0.1
.050 V
AooB E
5

8
2
0.01
5 10 15 20 0
1.0
Iz
t/2 = 5.40 7.15 7.89 14.77 16.72 33.47
> 0.5
T 37 34 30 26 20 10
0.1
> Fig. 5. Luminal alkalinization (decrease of luminal acid phosphate
0 concentration) in proximal tubule of isolated perfused rat kidney at
different temperatures (CC). t/2, alkalinization half—time. Data from [40].
0.01 — 0.01
5 10 15 20 5 10 15 20
Time, sec epithelium, showing the importance of membrane—spanning
Fig. 4. Analog electric model for proximal tubule H transport and molecules or of H ion carriers for the passive flow of these ions
simulation of variation of protonmotive force (E), series resistance of [38].
the pump (RH) and luminal capacitance (C) with time. Effect of In other studies, the temperature of the perfusing solution of
variation of shunt resistance (R5) is similar to that of RH. Modified from
[39]. the isolated rat kidney was reduced progressively [41]. The
rates of both H ion secretion and back—flux were also reduced;
kinetically this effect was due to the increase in both acidifica-
tion and alkalinization half—times. Figure 5 summarizes the
the isolated—perfused rat kidney than in in vivo situation, but relation between half—times and temperature. Note that al-
these values are of the same order of magnitude as those though the rate at which H ions are transferred decreases with
obtained by Schwartz [30] and by Hamm et al [27]. As ex- temperature, the stationary transepithelial H gradient is much
pected, epithelial permeability to H is reduced in the lower less affected. Activation energies for these processes are ob-
temperature range. Apparent H is proportionately reduced in tained by drawing Arrhenius—plots using alkalinization and
low Na medium. The substitution of sodium by choline and acidification rate coefficients (k = in 2/t 1/2), 8.48 kcal/mol for
addition of amiloride causes an increase in alkalinization half- acidification and 9.31 kcal/mol alkalinization. These values are
time (from 6.3 to 22.6 sec) and a modification of the stationary of the same order of magnitude, indicating that movement of
transepithelial-pH gradient which is virtually abolished. This, H across the luminal membrane of proximal renal tubules is
according to an analog model for renal tubule acidification (Fig. symmetrical both from a kinetic (similar acidification and
4), corresponds to either an increase in pump series or shunt aikalinization half—times at a given temperature) and from an
resistance. An increase in these resistances corresponds to a energetic point of view. Thus, these studies support the notion
slower decay of CV, the luminal H-ion charge (C = capacity, V that H ion transport in both directions proceeds via a similar (or
voltage) shown in the graphs of Figure 4 [39]. Thus, these the same) mechanism, which appears to be the Na/H ex-
results suggest that the Na/H exchanger may participate in H changer.
ion back—flow from proximal tubular lumen. The nature of the In conclusion, H ion permeability of the luminal membrane of
remaining H ion flux out of the lumen is not clearly established proximal tubule does not depend only on flow through the Na/H
by this experiment. If Na/H exchange were completely abol- exchanger. A finite value for a parallel shunt path exists, higher
ished by these procedures, the remaining flux could be attrib- than that for other ion species, but lower than that measured
uted to passive flow across a transmembrane shunt path, and when the gradients for Na and H are reversed (as compared to
the corresponding H would represent non-mediated permeabil- the physiological situation). When luminal pH is lowered below
ity of this membrane (Table 1). the physiological stationary situation the Na/H exchanger con-
Work by Ives and Ives and Verkman on brush—border tributes to H ion back—flux, yielding a more elevated apparent
membrane vesicles, evaluating H ion permeability by acri- permeability than when the exchanger is excluded or set in the
dine—orange gradient dissipation, succeeded in measuring PH opposite direction.
independent of the Na/H exchange system [28, 40]. Their A protonophore incorporated into tubule cell membranes can
measurements yielded values of the order of 5 x i0 cm/sec enhance the role of an H ion back—leak. We have demonstrated
and were not affected by heat inactivation of the Na/H ex- that proximal tubules perfused with iO M dinitrophenol
changer; they also were insensitive to amiloride. These authors showed markedly enhanced W efflux from the lumen [42], and
believe, on the basis of these studies, that H/OH back—flux in that the incorporation of amphotericin B, a polyene antibiotic,
brush border membranes does not occur by "slippage" of the into distal tubule luminal membranes has a similar effect [43]. In
Na/H exchanger, but proceeds through a lipid pathway. The H both cases, stationary pH levels increase at a faster rate which
ion permeability of lipid bilayer membranes, on the other hand, results in a reduced capacity to establish transepithelial pH
is about three orders of magnitude lower than that of the whole gradients.
140 Malnic

Lumen Capillary
) Ju, nmol/cm sec
0
Active HCO3(H) ' 3.33

cc
E HC03 leak 0.15
0
0.
H leak 0.004
cc
uJ
10
3 17
8

pH = 7.13, HC03 = 19.5mM

Blood:
0
C.)
C.,

pH = 7.3 HC03 = 28.5mM

Active HCO3_(H*) 0.42

S
0cc HCO3 leak •:: 0.38
0)
0,
C
H leak 0.04
E
-J
Net HC03 0 5 10 15 20 25
Time, sec
Fig. 7. Kinetics of proximal tubule bicarbonate reabsorption in control
pH = 6.6, HC03 = 5.7 m
(0, t/2 = 3.8s) and Diamox (I, t/2 = 8.ls) infused rats. Log of
bicarbonate concentrations (at time t minus stationary levels) is plotted
against time. Modified from [331.
Fig. 6. Components of acid,Yi cation in early and late proximal tubule.
Fluxes of the active and passive components and early and late
proximal luminal pH and HC03 are given. From [44].
proximal tubule. In acute alkalosis due to bicarbonate infusion,
the absolute proximal reabsorption of this anion is increased
A word on the physiological role of H ion back—flux is now in [45, 46].
order. The effective role of such fluxes depends on H perme- We have used stopped—flow microperfusion experiments to
ability, which is very high whichever of the described values is demonstrate in a more direct way how luminal bicarbonate
accepted, and on the driving concentration, which is below concentration determines its rate of reabsorption. Using this
micromolar in biological media. The net result of these factors technique, summarized in Figure 2, we injected solutions con-
is very low net back—flux in normal or near—normal conditions. taining between 25 and 100 mri bicarbonate into the tubular
Figure 6 gives a schematic view of the main components of the lumen. The concentration of this anion reduced exponentially,
proximal—tubule acidification mechanism as proposed by Chan, toward its stationary level. In Figure 7 the semilog plot shown
Malnic and Giebisch [441. Clearly, the role of passive H yields a straight line [33] from the initial level of about 50 mrs'i
back—flux is minor even in the upper pH range of the reported down to the stationary level (3 to 6 m in control proximal
data, and passive bicarbonate leak represents a larger portion of tubule). This graph indicates that the rate of disappearance of
the acidification shunt path. Furthermore, evidence favors a bicarbonate from the lumen, that is, the rate of bicarbonate
transcellular route of H and a paracellular path for HC03. reabsorption (JHco3) is, at every point of the line, proportional
The combined effect of these leaks is still too low to account for to the lumen concentration of the anion:
a constant pump—variable leak system [26]. Therefore, the
JHCO3 = —d(HC03)/dt = K (HC03) (1)
variation of the magnitude of net H-flux in the secretory
direction at different luminal pH points to a gradient—dependent where K is the rate coefficient of bicarbonate disappearance
Na/H exchange as the main operation of acid secretion. from the lumen, related to the half—time of this disappearance
by the relation:
Load dependence of renal tubule acidification
Since an increased filtered load of buffer enhances the rate of K=1n2/t/2 (2)
acidification by a given tubule segment, an increased filtered Thus, within the observed range of concentrations of luminal
load of bicarbonate enhances bicarbonate reabsorption by the bicarbonate, its reabsorption is linearly related to its load.
 H secretion in renal cortical tubules 141

300 Net
250

'1 200 E
E 200
0
E
150
E
0 00
E
100
100
0
I
U 0
50 LI

0
0 200 400 600 800 1000 0 10 20 30 40 50 60 70 80 90
Bicarbonate load, pmolmin' Mean luminal (total C02], (mM)
Fig. 8. Bicarbonate reabsorption along proximal convoluted tubule as Fig. 9. Total CO2 reabsorption by proximal convoluted tubule as
function of bicarbonate load. Symbols are: ( • ) vary perfusate function of luminal total CO2 level. Evaluation of proton secretion and
[HC03], constant peritubular pH; ( ) vary perfusion rate, tubular passive back—flux. From [52].
[HC03]: 25 mEq/liter. From [47].

An extensive study by Chan, Biagi and Giebisch [47] on single nephron GFR (SNGFR) values below normal values; at
several properties of proximal tubule bicarbonate—transport higher SNGFR saturation of bicarbonate reabsorption oc-
also showed a linear relation between lumen bicarbonate con- curred. They concluded that a reduced GFR was an important
centration and its reabsorption rate up to a luminal level of 44 factor in maintaining chronic metabolic alkalosis. On the other
m. Also, bicarbonate reabsorption showed a much steeper hand, Maddox and Gennari [56] found load—dependence in
relation with luminal bicarbonate concentration than with an chronic metabolic alkalosis to be similar to that in control rats,
increase of load effected via higher luminal flow rates (Fig. 8). so that the increased reabsorption of the bicarbonate load
In the early clearance studies, load dependence was observed maintains this condition. The differences in experimental data
up to an apparent Tm, corresponding to a filtered load not much and their interpretation may be due to different ranges of
higher than the normal filtered amount of bicarbonate [48, 49]. SNGFR and thus filtered load used in these investigations.
This apparent Tm was due to the expansion of extracellular Load dependence of H secretion occurs not only for bicar-
fluid volume caused by the infusion of the bicarbonate—contain- bonate reabsorption, but also for H ion secretion into other
ing solution. It was not due to transport saturation, but to the species of luminal buffers, whatever their nature. We have
paracellular back—flux of this anion due to peritubular Starling shown that the approach of luminal buffer concentrations to
forces modified by extracellular fluid volume expansion [45, 50, their stationary levels follows an exponential time course for
51]. However, microperfusion studies by Alpern, Cogan and different buffer systems [57]. Figure 10 shows this behavior for
Rector [52], in which luminal bicarbonate concentrations were the buffers glycodiazine and DM0. The rate of acidification of
even more elevated, showed that overall bicarbonate reabsorp- these buffers depends not only on the acidification properties of
tion saturated at luminal concentrations of about 60 m. These the tubular epithelium, but also on the permeability of this
authors evaluated the active (H secretion) and passive compo- epithelium to the buffer components. Buffer systems with
nents of bicarbonate reabsorption under these conditions (Fig. permeant components like CO2-H2C03 or undissociated glyco-
9), and found that H ion secretion saturated already at 45 mis'i diazine are reabsorbed at much higher rates than systems with
luminal bicarbonate. impermeant components like phosphate. In the latter systems
Load dependence of bicarbonate reabsorption also occurs in luminal pH drops faster than in the former since the acid,
both acute and chronic metabolic alkalosis. With the described titrated form remains within the lumen. If the acid component is
kinetic method, we observed bicarbonate reabsorption to be highly permeant it will be passively transferred to blood as soon
proportional to luminal concentrations. Although the capacity as it is generated in the lumen, and thus reabsorbed. These
to maintain transepithelial pH gradients as well as the reabsorp- experiments suggest that the determinant factor of load depen-
tion rate coefficient k were reduced in metabolic alkalosis, dence is luminal pH and not kinetic interaction of a buffer
bicarbonate reabsorption was still load dependent. Since component (such as bicarbonate) with a membrane site.
luminal HC03 levels are higher in these conditions, net In order to obtain more direct evidence for the role of luminal
reabsorption of the anion increased [53, 54]. Saturation of pH itself in determining rates of H ion secretion we applied the
bicarbonate transport is also present in this condition, although pH-stat method to renal tubules. This method has been exten-
the level at which saturation occurs varied in studies by sively applied to study epithelia like turtle bladders in vitro [22,
different authors. Cogan and Liu [55] found linear load depen- 58]. In this structure, a linear relation between mucosal pH and
dence only when the bicarbonate load in the rat was varied at secretory H-flux exists in the range from blood pH down to the
142 Malnic

10

0.5
E

0
c 0.2 0-J Fig. 11. Schematic representation of pH stat method. pH is measured
by a double—barrelled pH/reference electrode, and current (OH- or
HK) is injected under control of electronic feed—back system.

is the injection of base or acid via a point—source; as shown

schematically in Figure 11, the injected ions will be distributed
along the perfused segment in an unequal manner, similar to
what occurs when electrical current is injected. Although the
injected fluid is blocked between oil columns, pH drops from
the current injection point with distance. We can measure this
change to obtain a length constant for pH which is very similar
for OH- or W injection, of the order of 16 to 17 sm.
Consequently, we applied a method analogous to cable analysis
5 10 15 20 25 30
S
to calculate H fluxes under pH-stat conditions (pH-stat
Fig. 10. Kinetics of acidification of DM0 and glycodiazine in proximal fluxes), starting from the relation:
tubule. From [57J.
F5 = IW(pH1—pH) (3)
where I is the injected current, pH1 is the pH level maintained
maximal transepithelial pH gradient. As already indicated, H by the pH-stat system, pH the stationary pH level, and the
ion secretion in turtle bladder is a rheogenic, ATP-dependent effective pH-stat flux of H. From this value we can obtain a
process, a primary active—transport mechanism, as opposed to pH-stat flux (H ion flow per one pH unit) per cm2 of tubule
Na/H exchange, a secondary active—transport mechanism, in epithelium.
proximal tubule. Turtle bladder has, furthermore, very low Figure 12 summarizes effective pH-stat fluxes measured in
permeability to H ions, being a "tight" epithelium with low control conditions and after luminal perfusion with 104M
paracellular conductance. Due to these properties, H ion secre- acetazolamide. Several measurements were performed in se-
tion in bladder is gradient—dependent and falls to zero when the quence. Note that carbonic anhydrase inhibition reduces the
maximal transepithelial pH gradient is reached. The low passive amount of 0H injection needed to maintain a given change in
permeability of this structure makes it easy to study, since lumen pH. Since at a given pH the amount of OH- injected
experimentally measured rates of acidification represent mostly equals net epithelial H secretion, the reduction of 0H needed
unidirectional H-ion secretory fluxes. A similar method of reflects a reduced capacity of the epithelium to secrete H. This
studying acidification kinetics may also be applied to renal is expected after carbonic anhydrase inhibition. If a similar
tubules if we consider its geometry and specific properties. experiment is done while injecting H into the lumen, no
Figure 11 gives a schematic view of the pH-stat technique as reduction in effective pH-stat flux occurs. This is again reason-
applied by us to the renal tubule. A column of perfusion fluid (2 able, since under these conditions the H ions injected into the
to 5 mM phosphate Ringer's) is blocked with oil, pH is mea- lumen flow out across the epithelium; no cellular H-ion source
sured by means of a double—barreled antimony/reference micro- is needed for this H translocation.
electrode, and OH— or H ions are injected by passing current Figure 13 shows a graph constructed after converting effec-
through Sb or HC1-fflled microelectrodes. The magnitude of the tive pH-stat flux to flux per unit epithelial area and relating
injected current is regulated to maintain a predetermined pH these fluxes (JH) to the pH changes induced. This relation has
level either by an electronic feed—back circuit or by a digital an approximately linear portion at small pH changes, but as
computer programmed to keep luminal pH constant. A disad- these changes increase, there is less further modification in flux.
vantage of the application of the technique to tubular structures This phenomenon occurs both in the alkaline and in the acid
 H secretion in renal cortical tubules 143

30 1.0
JH/JH,,,ax 0
00

0
0
0.5

20

—2 —1 2
I I
pH

10
—0.5

0
0
0

I I I I a
—1.0
C C D D DD R R t Fig. 13. Normalized pH stat fluxes as a function of changes in luminal
pH above and below the stationary level.
Fig. 12. Effective pH stat fluxes in control proximal tubules and after
luminal perfusion with 1O M Diamox. R, recovery. Fluxes in
nanoamp. per pH unit.
rates may be regulated by the incorporation of pumps, stored in
vesicles in the apical cell region, into the luminal membrane.
direction, and is equivalent to the saturation of the H-ion For instance, stimulation of acidification by increased pCO2 has
transport system discussed above. When the luminal buffer load been shown to be mediated by vesicle exocytosis, both in
is increased, that is, when pH increases above a certain level, H amphibian bladder [60, 61] and in mammalian renal epithelium
ion secretion is not able to increase in proportion to this [62, 63]. Cell pH and calcium mediate exocytosis [61].
modification. Both in the alkaline and in the acid range, a During luminal bicarbonate loading, cell pH is alkalinized,
linearity occurs over a range of about 0.8 to 0.9 pH units above probably due to the increased rate of H secretion. O'Regan,
and below the stationary pH level.This corresponds, consider- Malnic and Giebisch [64] have directly demonstrated, by intra-
ing a normal luminal pH of 6.8, to a pH of 7.6 to 7.7, equivalent cellular pH measurement in Necturus proximal tubule, that
to a bicarbonate level between 40 to 50 m, near that observed bicarbonate loading reduces the electrochemical potential gra-
for saturation of bicarbonate transport. dient for H across the luminal membrane. Therefore, during
The general form of this graph also resembles the data buffer loading the increased H secretion is not mediated by an
obtained in turtle bladder. Steinmetz and Anderson [59] devel- increased number of membrane pumps. This view is also
oped a model of H ion transport for turtle bladder which also supported by our kinetic data, which show that the rate
shows saturation both at high and at low pH. Another property coefficient of H* secretion remains constant at different levels
of the bladder model is a symmetry of H ion fluxes, that is, of luminal bicarbonate. The changes in reabsorption rate at
these ions proceed through the pump in both directions, during these different luminal concentrations are instantaneous,
H secretion as well as during H back—flux. We have shown whereas the process of exocytosis is effective in a period of
evidence for similar behavior in the proximal tubule above. about one minute [62]. It thus appears that acute luminal pH
However, since the proximal tubule is a leaky epithelium, changes affect H secretion by modifying primarily the gradient
passive permeability to H, HC03 or to buffer H carriers all against which secretion occurs.
contribute to H back—flux via shunt pathways [26—28, 35].
Also, the turtle bladder model of Hion transport is not directly Role of the basolateral cell membrane in acidification
applicable to the proximal tubule due to the different nature of According to the acidification processes in renal epithelia
transport process (NaIH exchange) in the latter epithelium. summarized in Figure 1, H pump activity in the luminal cell
However, although differing in details, a proximal tubule model surface requires the transfer of base formed within the cellular
could be construed to have similar kinetic properties. There- compartment across the basolateral cell membrane. For a long
fore, we conclude that the H pumps of widely different nature time base extrusion was thought to be a simple passive process
and of different tissues share common properties of an EMF of bicarbonate (or OH—) diffusion along a favorable electro-
(apparent or effective proton—motive force) in series with a chemical potential gradient. Electrophysiological findings, by
resistance that limits the flow of H ions across the tissue [36]. Frömter and Sato, and Burckhardt, Sato and Frömter [65, 66]
An additional mechanism of how luminal pH regulates the initially supported this view. Figure 14 shows a record of the
transport rate of H has also been considered. H-ion secretory electrical potential changes measured across the basolateral cell
144 Malnic

—35 0 2 4 6 Bsec
HCO3-free I — I I
—40.
Control
—45,

Acz
—50
E

0 _55
SITS

—60

Gluconate
—65

—70
Ba

10 20 30 40 50 Capillary alkalinization t/2

Time, sec Fig. 15. Alkalinization half—times (t/2, sec) during stopped—flow
Fig. 14. Proximal tubule basolateral PD: Effect of bicarbonate—free microperfusion of peritubular capillaries in different experimental con-
peritubular perfusion. From [65]. ditions. Acz, acetazolamide. Data from [68].

membrane (BLM) by these workers when peritubular bicarbon- red blood cells, a mechanism which depends on the band-3
ate concentration is reduced at constant pH. The membrane protein component of the cell membrane [70, 71, 72]. Substitu-
immediately depolarized and a slower partial repolarization tion of chloride by gluconate also impaired base efflux from
follows; the former due to a bicarbonate diffusion potential and proximal cells. These experiments are compatible with the
the latter to the redistribution of cellular ions. Such potential existence of a chloride/bicarbonate exchanger in the basolateral
changes were markedly attenuated by acetazolamide and SITS. membrane. A similar transporter was reported for Necturus
Our laboratory used peritubular capillary perfusion and pH proximal tubule, but was also Nat-dependent and oriented in
determination to demonstrate that the efflux of base from renal the opposite direction and thus responsible for chloride reab-
epithelial cells is impaired by acetazolamide [67]. The effect of sorption [73]. Studies on cellular pH by the DM0 technique
carbonic anhydrase inhibition led to the view that the trans- reach similar conclusions [74, 75]. However, other evidence
ported ion species was OH-, the interconversion of this ion to does not support the presence of Cl/HC03 exchange in this
and from HC03 at the cytosollmembrane and membrane/in- tubular segment. For instance, changes in peritubular bicarbon-
terstitium interfaces being dependent on the activity of the ate concentration did not lead to alteration of intracellular
enzyme [66]. It was later found that the voltage changes caused chloride levels as determined by ion—sensitive resin microelec-
by reducing bicarbonate concentrations were also markedly trodes [76] or of cell pH as measured by a fluorescent dye [68].
depressed when sodium was absent from the peritubular me- In collecting duct cells evidence favors basolateral C1/HC03
dium, suggesting that bicarbonate efflux across the basolateral exchange [71, 72]. In this segment, a lumen—positive PD ap-
cell membrane was sodium—dependent [681. pears after inhibition of sodium transport, attributed to electro-
We used a stopped—flow microperfusion method in peritubu- genie H secretion. This potential difference was abolished by
lar capillaries to further study basolateral transport processes removing Cl from the bath and by SITS [77]. Similar findings
[69]. Peritubular capillaries where perfused with Ringer's solu- were made in turtle bladder [20, 78, 79].
tion containing 3 mri HC03 at 5% C02, at a pH of 6.6, and The reduction of basolateral base transport by barium ions
then blocked with colored light mineral—oil. We impaled a (Fig. 15) confirms that, as proposed by Frömter et a! [65, 66, 80]
perfused capillary with a pH microelectrode, and recorded the base transport at this site is electrogenic, since Ba blocks
return of capillary pH to the stationary level. This alkalinization basolateral potassium conductance and thus reduces the poten-
of the initially acid perfusion fluid is a measure of the rate of tial difference, one of the driving forces for base extrusion.
base extrusion across the basolateral cell membrane by the Boron and Boulpaep have shown in Ambystoma proximal
adjacent cells. Figure 15 summarizes the data obtained by this tubule that bicarbonate extrusion is electrogenic and sodium
procedure. The control alkalinization half—time of 2.33 0.12 dependent [81]. They concluded that changes of intracellular
sec increased significantly under a number of experimental H and Na activities during alteration of peritubular levels of
conditions. This increase indicates an impairment (delay) of the these ions were compatible with a single transport mechanism
base transport mechanism. The mechanism of acetazolamide carrying Nat, HCO3 and negative charge. A similar mechanism
action has been discussed above; SITS, as well as carbonic of coupling of one sodium to two or more bicarbonate ions, may
anhydrase inhibitors, block chloride/bicarbonate exchange in be operative at the basolateral membrane of mammalian kidney
 H secretion in renal cortical tubules 145

6.75 9.0 Phosphate Glycodiazine Phosphate

+ DIDS 104M
Phosphate

x0.
6.50f 7.0 C.,

('4
+ acetazolamide 104M

6.25 5.0

—' 1 mM 5mM 20mM 5mM 20 mivi 5mM 20mM 5 mM2O mM2O mM

6.00 [ 3.0
5mM 20mM 5mM 20mM
Peritubular buffer concentration
Fig. 16. Proximal tubule stationary pH and acidification half—time
during capillary perfusion with different buffer systems and at different
buffer concentrations. Symbols are: (0) HC03/C02; (Lx) HPO42/
H2P04; (0) GDZ/GDZH. From [95].
Peritubular buffer concentration
Fig. 17. Secretory H fluxes in proximal tubule during capillary perfu-
sion with different buffer solutions. Effect of buffer concentration,
DIDS and acetazolamide. From [951.
A
7.5
3

proximal tubules as well. Thus, Alpern [68] reported evidence —10 see—
for sodium dependence of basolateral base extrusion using
intracellular pH determination by a fluorometric technique.
Studies by Biagi and Sohtell [82, 83] on isolated, perfused rabbit
proximal—tubule demonstrated that the electrophysical re-
sponse to a low bicarbonate bath, indicative of bicarbonate IU
0.
effiux from the tubule cell, was delayed by SITS and inhibited
when sodium was substituted by tetramethylammonium
(TMA). Similar evidence was obtained in rabbit and Necturus
proximal tubule basolateral—membranes [84, 85]. Recently, a
3:1 HC03 to Na coupling ratio was postulated in rat and A B
rabbit proximal tubule [86, 87]
A number of investigations using basolateral membrane yes-
ides confirm that bicarbonate transport at this site is electro-
genic, that is, sensitive to transmembrane electrical potential —10 sec—

established by potassium ion gradients in the presence of

valinomycin. This process is also sodium dependent and Fig. 18. pH changes measured in peritubular capillaries during capil-
lary perfusion with 1 mr.t (upper) and 20 mM phosphate Ringer's
blocked by anion exchange inhibitors like SITS [88, 89]. Other (lower). A to B, luminal perfusion with pH 7.8 Ringer's solution. From
studies using the vesicle technique, based on uphill Cl uptake [95].
driven by HC03 gradients independent of PD [90], provide
evidence for an additional anion transporter such as Cl/HC03
or C1/anion exchange. A sodium—dependent anion—exchange
system with affinity for HC03 also exists at this site [91]. 68, 93]. An additional experimental evidence for such regulation
Our data summarized in Figure 15 are compatible with this is the role of peritubular buffer concentration in epithelial
view, since they support electrogenicity of bicarbonate transfer acidification. Studies on in vitro gastric mucosa and isolated
(shown by the effect of Ba* ) as well as carrier mediation of perfused rat kidney show elevation of rates of acidification
this process. A question that remains open is the role of when serosal or vascular perfusate buffer—concentrations are
chloride in basolateral bicarbonate transfer. The substitution of increased above physiological levels [58, 94].
chloride by gluconate leads to inhibition of base extrusion and We analyzed the relation between the nature of peritubular
is suggestive of chloride/bicarbonate coupling. Sasaki et al buffer and its concentration on acidification in rat doubly—per-
found similar evidence in rabbit proximal tubule, detecting an fused proximal tubule, in the expectation that this would give us
increase in cell pH after chloride substitution [84]. Sasaki and new insight on basolateral—membrane base transfer [95]. Ob-
Berry [92] observed inhibition of bicarbonate transport in this servations made in this study are summarized in Figure 16. At
epithelium when gluconate substituted for chloride, but attrib- higher peritubular buffer levels, whatever the nature of the
uted this to an unspecific effect of this anion, since this buffer, luminal stationary pH and acidification half—times tend
substitution also reduced transepithelial PD. Thus, there is now to decrease, reflecting elevated H-ion secretory rates (Fig. 17).
good evidence for Na(HC03) coupling, while an additional The effect of peritubular buffer concentration is abolished by
path of CY1HCO3 exchange needs further exploration. DIDS and by acetazolamide in capillary perfusate, indicating
The use of SITS or DIDS has helped clarify the role of that this buffer concentration effect depends on specific trans-
basolateral—membrane base transport in regulating renal tubular port of base. The mechanism by which these buffer levels may
acidification. Such drugs inhibit bicarbonate transport in the affect peritubular base transfer is shown in Figure 18. pH was
turtle bladder [20, 78, 79] and in mammalian renal tubules [66, measured in peritubular capillaries perfused with solutions at
146 Malnic
different buffer concentrations (1 and 20 m phosphate Ring-
er's). At the arrow, luminal perfusion with alkaline phosphate
(pH 7.8) was started. Note that when capillary buffer level is
low, capillary pH increases to a greater extent than when
capillary solution is more strongly buffered. Thus, a higher
buffer content avoids the establishment of an unfavorable pH
gradient for peritubular base extrusion and facilitates base
transport, which in turn increases transepithelial acidification.
These data underscore the important role of basolateral—
membrane base transfer for the maintenance and regulation of
overall acid transport across the proximal tubule.
Acidification in distal cortical tubules
The cortical distal tubule, the segment limited by the macula
densa and the junction with another tubule to form the collect-
ing duct, is characterized by cell heterogeneity. According to
Kriz and Kaissling [96] at least three different segments consti-
tute the so—called cortical distal tubule beyond the macula
densa: the distal convoluted tubule (DCT), the connecting
tubule (CNT) and the cortical collecting duct (CCD). Each of
these segments has a specific cell type, which in the case of the
CCD is the so—called principal cell. Additionally, intercalated
cells are found in both CCD and CNT. These cells are rich in
carbonic anhydrase and are present also in the cortical and
medullary collecting duct. They are akin to the carbonic—anhy-
drase—rich cells of turtle and toad bladder, and are, in all these
structures, thought to be responsible for H ion secretion [97,
98]. These segments differ not only in their morphological
aspects, but also in functional properties, as evidenced by the
work of Morel and collaborators, who have shown specific
binding of hormones, that is, specific receptors, in different
segments and cell types [99, 100]. This anatomical and func-
tional complexity makes it difficult to analyze the functional
properties of cortical distal tubule by micropuncture and
microperfusion techniques [101].
Early studies employing free—flow micropuncture showed
that distal luminal pH levels and derived bicarbonate concen-
trations are lower than late proximal values. Mean proximal pH
values ranging from 7.10 in early to 6.70 in late segments [44,
102, 103] compared to levels between 6.70 and 6.33 along the
distal tubule. Stationary pH measurements obtained in oil-
blocked segments show also more elevated pH levels in early
than in late distal segments. Thus, control rats exhibit a mean
early distal, stationary pH level of 6.95 and a late distal level of
6.60 [34]. These findings of accentuated acidification are com-
patible with the presence of intercalated, carbonic anhydrase—
rich cells more likely to occur in late than in early distal
segments. Thus, the terminal part of the distal tubule is likely to
be the site of an H transport mechanism similar to that
described for cortical collecting duct [211.
More recently, these findings were questioned by two groups
of investigators. In studies summarized in Figure 19, continu-
ous (pump) perfusions with bicarbonate containing solutions
were performed by Lucci et al [104]. Bicarbonate reabsorption
(as measured by microcalorimetric total CO2 determinations)
could not be detected in control rats, but only in animals
subjected to chronic metabolic acidosis. This was confirmed by
Levine [105]. These data suggest that no significant acid secre-
tion occurs in distal tubule in normal rats, but that secretion
could be induced by specific experimental conditions. Addi-
60
40
E
E
20
Fig. 19. Total CO2 fluxes in distal convoluted tubules microperfused in
vivo during different acid—base conditions. Symbols are: () control;
(11) respiratory acidosis, metabolic alkalosis; () respiratory acidosis;
(U) chronic metabolic acidosis; () acute metabolic acidosis. From
[104].
tional investigations involved measurements of distal pH and
pCO2 [103] and of disequilibrium pH by comparing in situ pH
with equilibrium pH [1061. No significant disequilibrium pH was
found in control rats, but a disequilibrium pH was induced in
acidotic conditions.
In the absence of distal bicarbonate reabsorption, a progres-
sive increase of pH along the segment would be expected since
significant fluid reabsorption occurs along this segment, mea-
sured by the progression of inulin TF/P ratios. This question
was recently reinvestigated by Capasso et al [107] by measuring
total CO2 and inulin levels along the distal tubule in control rats
(Fig. 20). While inulin TF/P ratios increase from about 3 to 9
along this segment, no significant elevation of bicarbonate
concentrations occurs; this corresponds to a progressive de-
crease of bicarbonate/inulin TF/P ratios, that is, to net bicar-
bonate reabsorption. The reason for the discrepancy with the
pump perfusion experiments cited above is not entirely clear. A
possibility would be that during pump perfusion some luminal
component (such as ammonia) present in free—flow conditions
might be absent; fluid reabsorption is also considerably higher
in the free—flow situation, which may reflect a different func-
tional situation of the segment.
Capasso et al also showed that in potassium depleted rats
distal fractional bicarbonate—reabsorption is enhanced. Also,
when such rats were given bicarbonate, the distal bicarbonate
load and absolute reabsorption were significantly stimulated.
Thus, distal bicarbonate reabsorption is load—dependent [1071.
The presence of distal H ion secretion is also supported by
a series of studies from our laboratory in which transepithelial
pH gradients were assessed directly. pH measured in stationary
columns of fluid within the tubular lumen was compared to
peritubular capillary pH, both measured by double—barreled
Sb/reference microelectrodes or by single—barreled pH-resin
 H secretion in renal cortical tubules 147

20

10
HC03/B HCO3 IB
Sb Resin
0.8
c.
U-
F-

2 0.6

20 40 60 80 100 0.4

0.2
E L E L

0.05

I I I I I
20 40 60 80 100 P04/B P04/P04
0.2 Sb Sb
0.6
0.1

0.05
0.4
0
0.01

0.005 0.2
E L E L

I I I I I
0 20 40 60 80 100
% Distal
Fig. 20. Bicarbonate and inulin ratios along distal convoluted tubules
in free—flow micropuncture experiments in control rats. Shaded area
represents range of distribution of measurements. Data from [1071.
and Ling—Gerard microelectrodes (Lopes, Reboucas and
Malnic, unpublished results). This technique has the advantage
of avoiding reliance on absolute pH determination and allows
direct measurement of pH differences with the same rnicroelec-
trode across the tubular epithelium. A summary of these studies
is given in Figure 21. Sizeable pH gradients across distal tubule
epithelium, being statistically significant in both early and late
segments, occur both during perfusions with bicarbonate and
phosphate-buffered solutions. The measured gradients are
markedly larger in late than in early segments, with both types
of electrodes used. These studies, as well as those by Capasso
et al, indicate that distal tubules of control rats possess a made in understanding the cellular mechanisms of urinary
significant capacity to acidify the urine.
The mechanism of distal tubule acidification is not entirely
clear. When comparing transepithelial bicarbonate gradients
and transepithelial PD, the electrical gradient is of sufficient
magnitude to account for the observed bicarbonate gradient. As
to the presence of H secretion, preliminary data obtained by
Reboucas and Cassola show that the distal transepithelial PD is
0.
Fig. 21. Mean pH differences measured with the same microelectrode
between lumen and peritubular capillary in distal convoluted tubules of
control rats. Stationary (oil block) conditions. E, early segments; L,
late segments. HCO3 /B, luminal perfusion with bicarbonate Ringer's,
blood in capillaries; P04, 20 m phosphate Ringer's; Sb, antimony pH
microelectrodes; resin, pH-exchanger resin microelectrodes.
affected in a very heterogeneous manner during luminal perfu-
sion with lO- to lO— M amiloride. In some segments no effect
of amiloride on PD was seen; in others, transepithelial PD fell to
zero, and in a third, late distal group, there was an inversion of
PD to lumen—positive values. The latter finding could be attrib-
uted to a segment with electrogenic H secretion as described
for cortical collecting ducts [211. These findings again stress the
functional heterogeneity of cortical distal tubules.
This presentation has shown the great progress research has
acidification. A question we should ask now is, what direction
will research take in the next years? Microelectrode and indi-
cator techniques for luminal and intracellular pH determination,
and the use of membrane vesicles will bring important new
insight into the mechanisms of H ion secretion. These tech-
niques will allow us a better understanding of the regulation of
cell pH and the mechanism of acid and base transfer across
148 Malnic
individual cell membranes. We can not only study in vivo and
isolated tubules, but also, with increasing frequency, cell cul-
tures in vitro, allowing us to study isolated cells of standardized
and well defined nature. Whether it will be possible to study
individual channels for H ion transfer is unknown due to the low
concentration of these ions in biological media.
We can also expect progress in the knowledge of mechanisms
of regulation of H ion transport, both by the study of molecular
mechanisms of interaction of H ions with carrier molecules, and
by methods of cell biology, which have led to the recent
knowledge of the role of membrane vesicle exocytosis in
regulating the rate of W transport across cell membranes.
Thus, knowledge on the cellular and molecular mechanisms of
acid secretion and its regulation should be greatly expanded.
Acknowledgments
Work in the author's laboratory has been supported by FAPESP,
CNPq, and FINEP. 1 thank Drs. A.C. Cassola, A.G. Lopes and G.
Giebisch for comments and revision of the manuscript.
Reprint requests to Gerhard Malnic, M.D., Department ofPhysiol-
ogy and Biophysics, Institute Ciencias Biomedicas, Universidad de São
Paulo, CP 4365, 01051 São Paulo, Brazil.
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