Kidney International, Vol. 9 (1976) p. /34—148

Renal tubular mechanisms of organic solute transport KARL J. ULLRICI-I Max-Planck-Insiitui für Biophysik, Frankfurt/Main, West Germany

Until recently the kidney was not among the organs favored for research on the molecular mecha-

one to measure the specificity and even kinetics of the overall transtubular organic solute transport and can

nisms of transport processes. The key experiments for

give a strong indication whether there is dependence on other transport processes, such as that of Na or H. The same method can also be used for electrical measurements [11]. Provided that the re-

exploring the molecular events of sugar and amino acid transport, for instance, were performed on intestinal [1, 2] and Ehrlich ascites tumor cells [3, 4]. The interest of the scientist studying molecular trans-

spective organic solute transport causes a change in the electrical potential difference, it is even possible to localize the event to either cell side by introducing an intracellular electrode.

port in the kidney rose rapidly, however, since the organ served as a source for transport enzymes such as

the Na-K-ATPase [5] or the glucose binding pro-

The main parameter measured in connection with the organic solute transport is the steady state electro-chemical potential difference, , at zero net flux of solutes and water. Under these conditions active transport, Jaet,1 equals passive backflux, the latter being proportional to the permeability coefficient, P,

tein [6] and especially since it became possible to obtain closed vesicles from either cell side of the proximal tubule [7, 8]. With these vesicles the transcellular transport steps could be studied separately without interference by cellular metabolism. Previously or simultaneously performed microperfusion [9, 10] and electrophysiological studies [11, 12] served as a basis for, or were at least complementary to, experiments with tubular membrane vesicles, At once some sim-

times the electrochemical potential difference, is the sum of the transtubular concentration difference, c, and the electrical potential difference (ii) which is brought to the proper dimension by multiplying with zF/RT and the mean transtubular concentration ë1. The equation J5 = P• (zc + zF/RT c1 is a measure of the active transz1i) shows that can port rate provided that P remains constant.

ilarities with the corresponding intestinal transport

processes became apparent [13]. Although our knowledge is growing rapidly so that many more an-

swers can be expected in the near future, the main principle for transtubular hexose and amino acid transport is already evident: co-transport with sodium (secondary active transport) at one cell side.

be measured directly or it can be evaluated by the dis-

tribution of an electrolyte which is not transported actively and for which the equation —' = c RT/ zF holds. zit' vanishes in the case of nonelectrolytes,

The transport step at the other cell side proceeds also by a carrier, but with a different specificity.

and is otherwise small across the proximal con-

The situation with organic acids, if they are not transported by nonionic diffusion, and with the or-

volution so that it can be neglected, especially ifc is large and if the electrolyte is univalent or only partially dissociated. Since almost all c measurements of organic solutes are made with isotopes instead of being based on chemical analyses, it is important that the measurements be performed when the net flux of

ganic bases is less clear, but a similar principle, possibly a whole chain of countertransport processes, may also hold for them. Because in this review, results gained largely from double-perfused kidney tubules and from membrane vesicles are reported, the corresponding methods and their potency should be discussed. The method of luminal, combined with simultaneous peritubular, capillary microperfusion [9] allows

isotope is also zero, that is, when the specific activities

in the luminal and capillary perfusate are the same. Another prerequisite is that the substance must not Here it has not been discriminated whether the transport is primarily active, i.e. directly driven by metabolic energy or secondarily active, i.e., coupled to transport of another substance which itself is transported by primarily active transport.

© 1976, by the International Society of Nephrology. Published by Springer-Verlag New York 134

135

Organic solute transport

be metabolized or that its metabolic rate must be small compared with its active transport rate. For electrical measurements, the concentration of the organic solutes within the luminal or peritubular perfusate has to be changed rapidly [14, l4a]. For this reason double or multibarrelled perfusion pipettes are used. On evaluating the potential changes across either cell side, it is important to realize that a potential change caused solely across one cell side will also show up at the other cell side because of the large par-

acellular shunt conductance [14, 14a]. This large shunt conductance is also the reason why consider-

hexoses [7, 13]. A third important feature is whether the uptake is influenced by the transvesicular electrical potential difference, the sign and the magnitude of which can be varied at will [17]. The most common procedure for this is to use one cation with anions of different permeabilities or to increase significantly and selectively the permeability for some other ion also present in the solutions by adding ionophores,

such as the K ionophore valinomycin or the

ionophore monactin. With proper modification of the experimental procedures it is not too difficult

to discriminate the mode of transport that pre-

able changes in the intracellular potential as

vails for a given substance: simple diffusion, carrier-

measured across one cell side can hardly be seen with transtubular measurements. The most critical aspects of transport studies with membrane vesicles [15] are the purity of the vesicular preparation and the tightness of the vesicles. Further-

mediated transport, co-transport, countertransport or ATP-driven transport.

more, fast and complete removal of the incubation fluid without backleak of solutes already taken up by the vesicles is essential. The homogeneity of the brush

Although micropuncture experiments had shown that a small amount of glucose is also reabsorbed in the distal convolution and collecting ducts [18], ex-

border preparation from the proximal convolution

periments relating to the mode and specificity of

can readily be checked by determination of enzymes such as alkaline phosphatase and the disaccharidases which are located only in the brush border and not in other kidney membranes. A marker enzyme for the basolateral membrane of the proximal tubule with a specificity similar to that for the brush border, unfortunately, does not exist. The cortical membrane fraction which contains the Na-K-ATPase seems however, to include little Na-K-ATPase activity which

transport were performed only in the proximal convolution [9]. In proximal tubules of the rat, evaluation of the transtubular steady state concentration differences c, which in the case of nonelectrolyte is a measure of the active transport rate, gave the following sequence

is not located within the basolateral cell membranes of the proximal convolution [16]. This subject as well as the various separation procedures were re-

Transport of sugars

in decreasing order: D-glucose, 3—methyl-D—glycoside, a—methyl—D—glycoside, 6—deoxy—D—glucose, D—galactose, f3—methyl—D—galactose, 3—0—methyl—glucose, D—

The active transport of all these sugars, the configuration of which is shown in Fig. la is abolallose.

ished by 104M phlorrhizin. No active transport could

cently reviewed by Kinne [15]. So far, differential

be documented for the sugars shown in Fig. lb:

gradient ceritrifugation and additional free-flow electrophoresis seem to give the purest membrane preparations. The tightness of the vesicles for molecules of the size of hexoses is readily tested by demonstrating that the intravesicular space varies inversely with the

L—glucose, D—mannose, 2—deoxy—D—glucose, D—fructose, D—glucosamine, 3—deoxy—D—glucose, 2—deoxy—

concentration of these substances in the incubation medium [7, 8]. For removal of the extravesicular test substances after incubation, the Millipore filter technique and washing with ice-cold Ringer's solution has proved to be satisfactory[7].

both was inhibited by other sugars in the order D—

The strongest indication for carrier-mediated transport through the vesicular membranes is the demonstration of an isotope counterfiux near equilibrium, i.e., the fact that preloading of the vesicles with

unlabelled substance enhances the uptake of the same, but labelled, compound. Furthermore, a carrier-mediated co-transport is indicated, if a mutual enhancement of the uptake of an electrolyte and a nonelectrolyte is observed, as in the case of Na and

D—galactose. Since a mutual inhibition of active transport was observed between o—methy1—D—glyco-

side and D—galactose and since the active transport of glucose > 6—deoxy—D—glucose > 3—0—methyl--glu-

cose, while D—mannose had no effect, one may infer that there is only one system for active hexose trans-

port. This system requires an OH group in the d-gluco configuration on C atom—2, a configuration which was found by Crane [19] for intestinal hexose transport as early as 1961. All deviations from this steric arrangement on C atom—2 are clearly excluded from active transport: D—manflose, 2—deoxy—D—glucose, D—fructose, 2—deoxy—galactose and D—glucosamine: A deviation from the D—glucose configuration

on C atom—3 permits a small active transport (3—0— methyl—D—glucose, D—allose) but not if the OH on

UI/rich

136 o —GJueose

f3—Mcthyl—D —glucoside

(6) CH, Oil H /fO\ (I)

a)

(4)

6—Deoxy—D —glucose

II

3—0—Methyl—u —g!uiose Cl 12 OH

CH3

0

H

HHCIl

H41H0

OH

H

CH2OH CH3

HUH H

HO

o—MetIIyl—D —glucoside

CH,Oll

OH

3—M e thyl—o —gal actosid e

Cu2 Oil

H

I)

H, Oil

01-I

110 H

OH

H

ii

OH

Oil

D —Calactose

u —Allose

CH2OH

('H2 OH

li>

HO

OH

1\çl-ll/ II

OIl

HO

Oil

OH

OIl

Fig. 1. a, Sugars which are actively transported by the rat proximal convolution. b, 0—NI all nose

2—Deoxy—o —glucose

I) —Glucosaiiiin

h)

Sugars which are not actively transported.

H Iii

H — C — 01-I

Il—C— OIl

H,,O\ll llO0 011 H

N\OH

110

Ii

H

11/Oil H

1)—Fructose

L —Glucose

3—Deoxy—u —glucose

(6) 110F12C

(2) H2OH

OIL

H

01-I H ()(l)

OH

HO

(4)

OH

1-1

C atom 3 is missing. If the deviation is on C atom

hyorogen bonds to it. In the intestinal mucosa a

4 (D—galactose), the active transport is nearly 70% of that of D—glUcose, and if the deviation is on C atom

similar N a -dependent D—glucose--D—galactose transport system was found, [20—22]. L—glucOSe, which was

I or 6 (— or

not actively transported in the experiments mentioned

3—methyl or 6—deoxy—D—glucose)2 the

transport rate is almost as large as that of D—glucose. The data suggest that the OH in the d—gluco—position

of C atom 2 forms a covalent bond with the carrier, while the OH's on C atom 3 and 4 apparently form

2

The transport rate of 6-deoxy-o-glucose was not measured directly but deduced from inhibition measurements and Its effect on luminal electrical potential differences.

here, was shown to be so in the rat proximal tubule under free-flow conditions by Baumanri and Huang [23], In these experiments a counter-transport of a reabsorptive D— with a secretory L—glucOSe move-

ment may have taken place. In the double-perfused kidney preparation it was shown that the c for all sugars transported actively dropped considerably when the perfusion solutions were Nat-free [9]. Detailed curves of c, i.e., active

137

Organic solute transport

transport rates, related to the luminal Na concentrations are presented for a—methyl—D--glycoside in Fig.

100

2. Three sets of experiments, each with constant

5 mmoles/liter

cs—methyl—D—glycoside in the perfusate but different

sodium concentrations, were performed. In each set, the active transport rate decreased with decreasing

16 mmoles/Iiter

0

luminal Na concentration and approached zero in a two-parameter Michaelis-Menten type fashion. The data allow a plot of the luminal a—methyl—

40

50 mmoles/liter

V

20

D—glycoside concentration against the correspond-

ing transport rate at different luminal sodium

a)

concentrations. The Km and Vmax(Lcmax) values cal-

culated for different luminal sodium concentrations showed that between 12.5 and 140 mEq/liter of Nat, the Km decreased by a factor of 4.1, but that z.cmax remained practically constant. The data indicated, but so far do not prove, a Na sugar cotransport system of the affinity type, i.e., where the binding of either Na or glucose to the carrier augments the affinity for the other ligand, leaving the mobility of the carrier-sodium-glucose complex within the membrane unchanged. This does not appear to hold for all species for both the renal and intestinal sugar transport [24]. Binding studies of phlorrhizin and its analogues to isolated brush border membranes give information about the first step of the glucose transport out of the tubular lumen, namely the binding to the brush border. Since D—glucose inhibits the phlorrhizin binding competitively, the K1 for D—glucose, which indicates its affinity toward its binding site, could be evaluated

[25, 26]. The most important findings of this study were that the phlorrhizin as well as the D—glucose binding were Nat—sensitive [27, 28]. The affinity of the receptor to either substrate, phlorrhizin as well as D—glucose, increased with rising ambient Na concentration with a KNa of 13 to 15 mEq/liter, which is almost identical with KNa for transtubular D—glucose

transport. Inhibition studies of phlorrhizin binding performed in another laboratory [29] gave specificity patterns which were, so far, in agreement with the transtubular transport studies mentioned above since the actively transported sugars D—glucose, D—galactose and 3—O—methyl—D—glucose inhibited

phlorrhizin binding, while sugars that are not actively transported, D—mannose, D—fructose, D—glucosamin and L—glucose, did not. The findings were, however, at variance in that 2—deoxy—D—glucose which is not

actively transported inhibited phlorrhizin binding, while the cs- and fl-methylglycosides, which were

readily transported actively, did not influence phlorrhizin binding. The reason for this discrepancy is not clear as yet. There is, on the other hand, a very nice parallelism between the sequence of the active

0

L_L

0

I

I

I

20 40 0 80 100 20 140 )IIL0//it'r Na

I

0

b)

2

3

4

5

ci Fig. 2. a, Zero net flux transtubular concentration difference (Zc) of 1C-a-methyl-o-g1ycoside plotted against the intratubular Na concentration. The chemical concentration of a-methyl-D-glucose con-

centration in the peritubular blood and therefore the maximal possible c (when ciumen is zero) were 50, 16 and 5 mmoles/liter [91. b, c vs. ic/c, plot ofa-methyl-D-glycoside for different luminal sodium concentrations. Using the curves of Fig. 2a the transtubular (sc) and luminal (c,) a-methyl-D-glycoside concentrations were read at 12.5, 25 and 140 mEq/liter luminal sodium concentrations and plotted according to Eadie and Hofstee. The line connecting these points was calculated by the Wang program 1047 A/GS2. The .c of the ordinate intercept corresponds to the LCmaX = prop. Vma. These values divided by the abscissa intercepts give the corresponding Km values [9].

transport rates [9], D—glucose > D—galactose > 3—0—methyl—glucose, and the potency of the phlorrhi-

zin analogues in inhibiting glucose transport: phloretin 2'—glucoside (phlorrhizin) > phioretin 2'—galactoside> phloretin 2'—(3—methoxyglucoside) [30]. These

data indicate that the differences in the active transport rates of these sugars were determined by their respective affinity towards the carrier. This is also supported by the fact that Na augments affinity as well as transport rate. The transport step of D—glucose through the brush border membrane of kidney cells in situ can be readily

138

Ui/rich

followed by measuring the transtubular or intracellular electrical potential differences [12, l4a, 311.

If the tubular lumen is perfused with the solutions containing D—glucose plus Na ions, then the transepithelial (lumen-positive) as well as the cellular electrical potential difference is reduced (Fig. 3a). The explanation for this phenomenon is that D—glucose

affects the electrical potential difference measured either between lumen and interstitium or between cell and interstitium. The glucose-coupled sodium influx from the lumen into the cell occurs along a combined

electrical and concentration gradient for Na1- ions. This movement is passive and the gradient is main-

crosses the brush border membrane together with

tained by the Na-K-ATPase which pumps the Na ions out of the cell at other, the contralumi-

Na1- in a charged Nat—glucose—carrier complex. This

nal or basolateral cell side. Since the Na-K-ATPase-

exerts a depolarizing effect on the luminal cell potential, as, for instance, in a nerve cell membrane when the sodium channels were opened. The circuit in Fig. 3b demonstrates how this reduction of the electrical resistance at the luminal brush border membrane (Rm)

mediated transport is directly driven by ATP, it is considered to be a primary active transport,

0 —-10

—20 —30 —40 —50

60

a)

Lu IIII

7

''U 20

s

1

I ntc Nti ti LI Ill

while the glucose transport through the brush border is a coupled secondary active transport. The specificity of the sugars which depolarized tubu-

lar cells so far coincides with the specificity of the

sugars which produce' a c: D-glucose, a- and fimethyl-glycoside 6-deoxy-D-glucose, D-galactose and

3-methyl-galactoside, depolarized, but D mannose and 2-deoxy-D-glucose did not [121 (Frömter E, unpublished data). A discrepancy was seen only with 3O-methyl-D-glucose which showed a small zc, i.e.,

active transport rate, which might, however, have been too small to be detected in the transtubular electrical potential measurements. With the electrical

measurements it was even possible to measure the kinetics of the transluminal D-glucose transport. Maruyama and Hoshi [32], found a Km of 1.15 mM for the newt kidney and Frömter and Lüer [12], a value of 1 mrvt for the rat, the latter being very close to the Km for the transtubular transport of D-glucose as determined in previous microperfusion experiments

[11]. From the D-glucose-induced changes of the electrical potential difference and the corresponding electrical resistance values, Frömter and Ltier [12] calculated the maximum electrical current which is induced by the Na1--D-glucose cotransport and compared it with the maximal D-glucose transport rates. b)

They arrived at equal flux rates suggesting a 1:

coupling between D-glucose and Na1- in the luminal Fig. 3. a, Electrical potential difference across the basal (peritubular) cotransport system. Since the coupled Na1--glucose transport is "electrogenic", it is sensitive to changes cell membrane of a proximal tubule cell of rat kidney when the lumen is perfused with a solution containing u-glucose. Abscissa = time, ordinate = electrical potential difference in my. At P the tubular cell was impaled with the electrode. The electrical potential difference was measured across the peritubular cell membrane. First the lumen was perfused with a HCO3- Ringer's solution; during the time marked by the bar G this solution contained 5 mmoles/liter of u-glucose. The fast depolarization of the luminal and peritubular cell membrane by u-glucose is explained by the electrical circuit depicted in b. b, R,,, Rb and RSh,,,, are electrical resistances of the luminal, contraluminal cell membranes and paracellular shunt pathway. respectively, u-glucose increases the passive entrance of Na into the cell (J1). Since both cell membranes in connection with the shunt pathway form a closed circuit, it is possible that after

having passed through Rm, current flows not only through Rb

which depolarizes the peritubular cell membrane but also through R,hUt,, rendering the lumen negative with respect to the

interstitial fluid [l4al.

in the transcellular electrical potential difference. On the other hand, by the changes of the electrical potential difference which it produces, it itself influences all other electrogenic co- or countertransport processes and also the passive transport of the electrolytes.

The mutual interaction between D-glucose-and

Na fluxes is very elegantly documented by flux studies

on closed vesicles of brush border membranes [7]. Whether the vesicles are incubated in a sodium-free solution or in the presence of phlorrhizin, the same 1)-glucose influx rate is observed until equilibrium is reached (Fig. 4). This influx could be attributed to a

139

Organic solute transport

passive, probably not carrier-mediated, influx into the vesicles. If, however, Na is added to the incuba-

2.5

tion medium, then the primary influx of D-glucose is

accelerated by up to ten times and, transiently, a

100 'ii Eq of NaCI

2.0

D-glucose concentration is reached within the vesicles

which is larger than the equilibrium concentration. This "overshot" of the intravesicular glucose con-

centration could easily be explained by Na ions moving from the incubation medium into the vesicles carrying 1)-glucose via the common carrier in addition to the passive influx of glucose which is proportional

1.5

100 mEq ol NaCI (vesiclcs preloaded with sodin ml

1.0

0.5

to the concentration difference of glucose. If this is

the case, no overshoot should be observed if the vesicles are in equilibrium with the surrounding Na ions from the beginning of the D-glucose flux study. As shown in Fig. 4, this is indeed the case. The electrogenic mode of the Na-g1ucose transfer, as already discussed above, leads one to suppose that, by the

rapid movement of sodium ions into the vesicles together with D-glucoSe, the intravesicular space becomes electrically positive compared to the medium.

If the anions present cannot follow as fast, the electrical potential difference would retard further Naglucose influx. That this is indeed so is documented by the fact that with faster moving anions (SCN), the initial influx and overshot became larger, while with slowly moving SO ions the overshoot is abolished. An acceleration of the glucose influx with a larger overshoot should also be observed, if an intravesicular negative electrical potential difference could

be established [14] (Murer H, unpublished data). This was indeed achieved by preloading the vesicles with K in the presence of the K ionophore valino-

mycin which makes the vesicular wall extremely permeable to K. Alternatively the vesicles were preloaded with H ions in the presence of an uncoupler like carbonylcyanide m-cholorphenylhydrazone (CCCP) which makes the vesicular wall permeable

to H ions. All these data support the Na gradient hypothesis. While the D-glucose influx into brush border mem-

brane vesicles is Na*dependent and could be inhibited by phlorrhizin, but only to a small extent by its aglucone phloretin, the D-glucose uptake by basolateral membrane vesicles is almost completely Naindependent and could be inhibited rather by phloretin

than by phlorrhizin. This indicates that the transfer of 1)-glucose through the contraluminal membrane of the proximal tubular cell is also carrier-mediated [7], This system seems to possess a different specificity

which, however, is not yet clarified. The different Na -dependence and specificity of the hexose transport through the different cell sides would explain results

gained from hexose uptake studies in kidney slices

0

0

2

4

6

8

10

12

14

lncuba tion tinic, I/li,, Fig. 4. Glucose uptake in closed vesicles from the brush border of the

proximal renal tubule. Upper curve together with Nat, lower curve Na was given before glucose so that the vesicles were already in equilibrium with respect to Na [7].

which are in some points of variance with the data presented above [24, 33, 34]. It would also explain discrepancies with data from uptake studies into yesides, which were probably insufficiently separated according to their origin from the brush border or the basolateral cell side [35].

The evaluation of the active transport rate of the hexoses based on c values, as mentioned above, reflects only the active step of the transtubular transport, namely the Na-hexose cotransport through the brush border. The second step, the carrier-mediated passive exit of glucose from the cell into the interstitium, did not show up in this type of experiment. This is also the reason why the results gained either by c- or electrical measurements or by the uptake in electrophoretically separated brush border vesicles are in fairly good agreement. If, however, the two transtubular transport steps of the hexoses are not experimentally separated, one may find a mutual inhib-

ition between 1)-glucose and 2-deoxy-D-glucose transport [34, 36]. This, however, does not necessarily prove the existence of one single transport mechanism for both hexoses. The interaction between both hexoses might rather occur at the second, passive exit step of the hexoses from the cell into the

interstitium. If, however, as in experiments on the whole dog kidney, an active D-mannose transport is observed [36], which is not seen in the proximal con-

volution of the rat, then one has to consider, first, that this transport might occur in nephron segments located distally from the proximal convolution and, secondly, that species differences might exist. Renal transport of amino acids

Since the transport of amino acids is sodium-dependent and occurs as an electrogenic cotransport

140

U/Inch

with Nat, a mutual inhibition of amino acids in their transport does not necessarily indicate, as pointed out above, that they share a common transport system. Therefore, at the moment, the safest indication for the diverse amino acid transport systems seems to be data gained from individuals with genetic defects. This topic has been reviewed thoroughly by Scriver and Hechtman [37] as well as by Deetjen, Foulkes and

Silbernagl [38]. In some instances, however, the transport systems indicated by genetic defects could not unequivocally be proved by detailed transport studies although sophisticated interactions have been proposed [39, 40]. In these studies only parallel transport systems with different but overlapping specificities have been considered, although an in-series arrangement of such systems within the luminal and

contraluminal cell membrane is also possible. Such

in-series transport steps are indicated by transit curves from renal artery to vein or from artery to ureter [41, 42]. Furthermore, in patients with lysinuric

protein intolerance, a net secretion of two amino acids of the arginine, lysine, ornithine (ALO) trio was observed, when the plasma level of the third amino

acid was increased [40]. This indicates a countertransport which changes the direction of the transport from an active reabsorption to secretion. But again such phenomena could be explained satisfactorily only if more is known about specificity and transport characteristics of the amino acid transfer through either cell side. Despite this restriction, cornprehensive microperfusion studies form a good basis for further analysis. In one of these studies Silbernagi and Deetjen [43] determined the molecular specificity for transtubular L-arginine transport and found that

two amino groups, with more than 2 CH2 between

IOU

them, are essential for interacting with the L-arginine reabsorptive system. The w-amino group has to be io-

,

80

tIer

60

niiioles/liter 40

I mrnoles/Iiter

nized, but the carboxylic group seems to be dispensable. Furthermore, they suggested that L-argi0

nine

is reabsorbed by more than one transport

system.

Experiments with the double-microperfused rat

a)

0

kidney [10] revealed that the active transport through the main transport systems, proposed previously, is Nat-dependent. These systems are the one for neutral amino acids (phenylalanine, histidine, amino-bicy-

20 40 60 80 100 120 140

0

Na urn. iuEq/liler

cloheptan-carbocyclic acid, aminoisobutyric acid),

the system for basic amino acids (lysine, orni4 3

'.

'

.

05

10

.

b) 0

thine, arginine), the one for acidic amino acids (aspartic acid) and finally the iminoglycine system (proline, glycine). In this regard the kidney behaves like the intestine where the active transmural transport of all amino acids is also Nat-dependent [2]. In the absence of Nat, only a passive amino acid influx from

the intestinal lumen into the epithelial cell takes

• "1o, 1

.

20

2.5

place, supposedly by the same carrier which in the presence of a Na -gradient provides secondary active cotransport with Nat. A kinetic analysis [10] of the active L-ornithifle transport (c) in the proximal tu-

Fig. 5. a, Zero net flux transtubular concentration difference (ic) of

or 3H-L-ornithine plotted against the intraluminal Na

bule of the rat kidney (Fig. 5), which was made in the same manner as described above for -methyl-glyco-

concentration. The chemical concentration of i,-ornithine in the peritubular capillary perfusate and therefore the maximal possible \c (when ciume is zero) was 2, Sand 10 mmoles/liter [10]. b, .

side showed that the Na-ornithine cotransport is of . the mixed type [45], Na ions not only diminish Km,

c/c1 plot of L-ornithine for different luminal sodium concentrations.

but also augment Vm&x for L-ornithine which means

L-ornithine concentrations were read at 12.5, 25, and 140

that Na ions increase the affinity of the amino acid .

mEq/!iter luminal sodium concentrations and plotted according to the method of Eadie and Hofstee. The line connecting these points was calculated by the Wang program 1047 A/GS2. The L\c of the ordinate intercept corresponds to the cma. = prop. Vmx. These values divided by the abscissa intercepts give the corresponding Km

plex within the membrane as well. Recently the analysis of the amino acid transport by electrophysiological measurements and by trans-

Using the curves of Fig. 5a, the transtubular (Xc) and luminal (ce)

values [101.

.

toward its carrier and the mobility of the carrier corn.

port studies on kidney membrane vesicles has ad-

141

Organic solute transport

vanced almost as far as for sugar transport. It can therefore already be deduced from the data available

that the transport of the neutral as well as the dia-

minomonocarboxylic and the monoaminodicarboxylic amino acids proceeds through the brush border as electrogenic cotransport with Na ions. Samarzija, Frömter and Ge(3ner [46, 47] observed that L-phenylalanine, L-ornithine, L-lysine, L-asparate and

L-glutamate added to the luminal perfusate depolarize the luminal cell side in the same manner as the actively transported hexoses do. Here it should be mentioned that previous microchemical studies of the

monoaminodicarboxylic acid distribution in the proximal tubule of the rat kidney indicated that the

Nat-dependent transport system of these amino acids is located in the brush border [48]. The electro-

physiologic data of Samarzija and Frömter [46], however, are at variance with the findings of Hoshi [49] in the newt kidney where only the neutral amino acids produce a Na1 -dependent depolarization, while L-lysine depolarizes in a Nat-independent manner and L-aspartate does not depolarize at all although the L-aspartate transport is Na-dependent. The explanation for this discrepancy is that in the rat kidney the transport of all amino acids proceeds with a surplus

brush border. The number of the systems transporting neutral amino acids into the cell and their specificity, however, remain to be clarified. As can be deduced from L-phenylalanine flux into vesicles from

basolateral plasma membranes of proximal tubules, the exit from the cell into the interstitium seems to proceed by a Nat-independent carrier mechanism [47]. The specificity of the peritubular amino acid uptake systems was also studied by Foulkes and

Gieske [42] in rabbits by analyzing A-V transit curves. These authors came to the conclusion that the

peritubular transport system resembles at least in part that observed in the luminal membrane. In their sensitivity to heavy metals, however, the peritubular systems differ from those in the brush border membrane. Recently it was proposed that the y-glutamyl cycle functions as one of the amino acid transport systems in certain mammalian cells [44]. The above-mentioned

amino acid Na cotransport systems within the renal brush border, however, seem to function independently of the y-glutamy1 cycle. One drawback of the y-glutamyl cycle- amino acid transport hypothesis is that theoretically three molecules of ATP are neces-

positive charge provided by cotransport Na ions,

sary for the transport of one molecule of amino acids. At the moment, however, it cannot be excluded that

while in the newt kidney this is only the case for neu-

some not yet defined renal amino acid transport

tral amino acids. The positively charged L-lysine might be driven through the brush border by its favorable electrochemical gradient, since L-aspartate may cross with just one Na ion in an electroneutral

might proceed via the y-glutamyl cycle. Since the amino acid transport in bacteria, yeast and mitochondria [51, 52, 53] occurs as cotransport with H ions, it is not improbable that the amino acid

ternary carrier complex.

transport across mammalian cell membranes pro-

The driving force for Na ions across the brush

border can be estimated to be around 120 my. If an amino acid depolarizes by 15 my, then the additional depolarizing effect of a second superimposed amino acid, which does not use the same transport system,

ceeds as cotransport with H ions as well. Such a system was proposed by Christensen et al [54, 55] for diamino acid uptake into Ehrlich cells. Experiments on

should be almost the same as when it was given alone.

If, however, the depolarization on superimposing a second amino acid is smaller than predicted, both amino acids must use the same transport systems. The electrical methods therefore can be used for specificity studies as was shown by Samarzija and Fromter [46]. In isolated brush border membrane vesicles, the influx of i-phenylalanine is Na -dependent [47]. Figure 6 shows an overshoot phenomenon depending on the

permeation of the accompanying anions and on an imposed electrical potential difference and provides a

countertransport with isotope-L-phenylalanine in a manner analogous to that of the hexoses described above. These data provide further evidence that the active transport of the neutral amino acids proceeds

as electrogenic cotransport with Na through the

60

'y

— Eq ui Ii6 ri tim co toe itt ratio it

I

ool ''S

0.16 s X 1 _9 i . mg ol prot.

(N=8)

Tinie. lit/il Fig. 6. Uptake of i-phenylalanine into closed vesicles from the brush border of the rat proximal tubule. Upper curve, together with Nat,

lower curve Na was added before t.-phenylalanine so that the vesicles were already in equilibrium with respect to Na [47].

142

Ullrich

the kidney should clarify whether this holds for this organ, too. Several important findings concerning the renal transport of D-amino acids were published in the recent years. For D-tryptophan, for instance, a net secretion was observed, which was proposed to occur as countertransport against the L-enantiomorph [50, 56]. In the double-perfused rat kidney, an active reabsorption of -histidine was observed which was 40% of that of L-histidine, while for D-alanine it was only in the range of 10% of that of the L-isomer [10]. Furthermore, in slices of human kidney a concentrative uptake of D-lySine was observed, which could be inhibited by L-lysine [57]. These findings are encour-

perfusion solutions were free of sodium.

aging for further electrophysiologic and micro-

mitochondria [81, 82]. In the kidney cell, too, a gradient for passive H flux into the cell and 0H flux out of the cell exists (83, 84]. Under normal

vesicular studies urgently needed to clarify the D-Lstereospecificity of the different amino acid transport systems. Transport of organic acids and bases

Organic acids and bases are actively transported in

the proximal tubule by at least three transport systems: The first is a rather unspecific secretory system

for organic acids with para-aminohippuric acid (PAH) as prototype. A second secretory and also unspecific system exists for organic bases with tetraethylammonium (TEA) as prototype and a reabsorptive system acting via H* ion secretion and nonionic diffusion.So far it is not yet clear whether uric acid is transported by a separate system or by that for organic acids [58, 59] and possibly also by nonionic diffusion, The transport of organic acids and bases has been critically reviewed by Weiner and Fanelli

[60, 61] and Rennik [62] and that of uric acid by Fanelli and Beyer [63] and Lassiter [64]. Although the methods of the double-perfused kidney and of flux measurement on kidney membrane vesicles were also applied to an investigation of the mechanism of PAH transport, the data so far available do not allow unequivocal conclusions. Nevertheless, they already restrict speculations. First we are faced with the question as to whether the active transport of organic acid proceeds by a primary active system driven by ATP

or by a secondary active Na (K) or H (0H) coor countertransport system. So far, there exist no

Furthermore, in PAH uptake studies into kidney membrane vesicles it was shown that Na and K, with varying anions, influence the PAH uptake by changing the transtubular electrical potential difference rather than by exerting a specific effect [80].

Therefore the Na-K-ouabain effect on the PAH transport seems to be secondary via another transport process. Since it was found that the renal H ion secretion is Nat-dependent and ouabain-sensitive,

one may suggest that the H (0H) transport provides the direct link to the transport of organic acids.

Such a countertransport of OH- and organic acids has been proposed for the organic acid uptake by

conditions the passive OH- outfiux from the cell into the interstitium, i.e., the site where the active PAH uptake occurs [80, 85, 86], is many times larger than the PAH uptake. Therefore, only a small part has to be coupled to drive the organic acid uptake into the cell. Furthermore, the first data gained on basolateral membrane vesicles under a pH gradient point to an

0H-PAH countertransport. By analogy with the substrate transport in mitochondria, a whole cascade of organic acid exchange processes may follow. If, furthermore, the transit of organic acids through the brush border is also carrier-mediated, a fact which remains to be proven, then it is not too difficult to explain why in the same tubular segment net secretion can be changed into net reabsorption [87]. The same may hold for uric acid transport too. A cascade of countertransport processes with different specific-

ity and mutual inhibition at the two cell sites may also explain the stimulatory or the stimulatory-inhibitory effect of acetate, lactate, pyruvate, citrate, etc. on PAH transport [88-95]. The suitable experimental procedures to test these possibilities are trans-

port studies on isolated kidney membrane vesicles from the two sides of proximal tubular cells. Transport of organic buffers by nonionic diffusion

If the nonionic component (AH) of a buffer is lipid-soluable and therefore readily permeant, then

experimental data which indicate a primary active transport system for PAH. But there are many findings which show that the PAH or phenol red transport depends on the presence of Na ions [65-69] and K ions [70-76] and can be inhibited by ouabain [73,

the buffer is transported via active H (OH-) ion

77, 78]. In the proximal tubule of the double-perfused rat kidney, however, only a small reduction of trans-

of 5.7 which is representative of the many long-acting

tubular PAH secretion was observed [79] when the

secretion and nonionic diffusion (Fig. 7). This topic has been extensively studied in the proximal tubule of the rat kidney [84, 96-98], but also on the perfused pancreas [99, 100]. Glycodiazine, a buffer with a pK

sulfonamides, is reabsorbed in the proximal convolution at the same rate as bicarbonate [98]. Its

143

Organic solute transport

transport, although not carbonic anhydrase-dependent, can be partially inhibited by acetazolamide. The active buffer reabsorption creates electrochemical

driving forces for the passive reabsorption of Na and C1 out of the proximal convolution [96]. Furthermore the H ion secretion within the brush bor-

der seems to proceed via an H-, Na

countertransport [98, 135]. Whether this counter-

—35

rHco31

—40 L —45 —50 H

transport is linked with ATP hydrolysis or synthesis remains open. A fascinating piece of evidence that nonionic and parallel H (OH-) transport also takes

place through the contraluminal cell side of the proximal tubule was provided by the electrical mea-

surements of Frömter and Sato [84]. If the pentubular blood capillaries are first perfused with NaC1

and then with a solution containing bicarbonate or one of the buffers glycodiazine, sulfamerazine or

butyrate, then the peritubular cell side is hyperpolarized (Fig. 8). This indicates that this cell side is highly permeable to the buffer anion A-. Since the anions are large and since it is unlikely that nature invented carrier systems for the chemically very different A-, it seems rather that the A- combined with H from water and penetrated as AH, in parallel with OH- (Fig. 7). Just after penetration A- and H20 may be formed again. In such a system all A- would penetrate the contraluminal cell side as AH and OH- and only one carrier for the facilitated diffusion of 0H would be necessary. Since it is not possible to dis-

criminate for the overall movement between 0H outfiux and H influx, a carrier-mediated H movement into the cell would serve the same function as a carrier-mediated OH- movement out of the cell. To what extent nonionic diffusion is involved in the transport of diverse organic acids and bases in different nephron segments where active H ion secretion

Cc!!

LLIIIIL II

A

In terstit LI III

l0,O

1120

Oil

011

—70

50 30 40 Time, sec Fig. & Effects of peritubular perfusion with HCO3-free solution on the cell membrane potential. Abscissa: time in seconds. Ordinate: potential difference across peritubular cell membrane in my, with suppression of zero value. Note typical potential response with 1) instantaneous depolarization on onset of perfusion, 2) rapid exponential-like repolarization, and 3) steady-state depolarization. Upon readmission of blood to the capillaries a mirror-image-like potential response is observed [84}.

0

10

20

occurs, namely the proximal and distal convolutions [101] and the collecting ducts [102] is unknown. The

studies of Swanson and Solomon in the pancreas show that even substances with a low pK such as acetate can readily be transported via nonionic diffusion [100]. Renal pinocytosis

Molecules below a diameter of - 30 A or a mol wt of 70,000 are readily filtered within the glomerulus,

but appear in the final urine only to a very small extent. They are pinocytosed within the proximal tubule. The renal resorptive pinocytosis, however, is only the special case of a general phenomenon [103105]. Since many peptide hormones such as insulin [106, 107], gastrin [108], glucagon [109], pituitary

ii

II

hormone [110], adrenocorticotropic hormone (ACTH) [111], parathyroid hormone [112] and growth hormone [113, 114] or toxins such as

+

+

staphylococcus enterotoxin B [115] are handled in

A—All

All

Fig. 7. Scheme of reabsorption of an anion (A-) by secretion of H and nonionic diffusion of the undissociated acid (A H).

this way by the kidney, much attention has been paid

to the process of renal pinocytosis within the last years. By direct microinjections of ferritin into kidney tubules and by taking electron microscopic pictures at different time intervals, the process of pinocytosis could be readily studied (Fig. 9) [116-120, 138]. Already two minutes after injection of ferritin into the tubular

144

Ullrich

containing particles and are transformed into lysosomes [119]. They contain, beside the pinocytosed

material, hydrolytic enzymes such as acid

Ill

phosphatase, /3-glucuronidase, -galactosidase, 13-Nacetylhexosaminidase, arylsulfatase [121, 122] and cathepsin A, B, and D [123]. Autophagic uptake of mitochondria, microbodies, etc. into the lysosomes was observed, too. The pino- and phagocytosed material is slowly degraded and the split products are released. The early steps of pinocytosis were also studied with isolated brush border membranes. Using the basic proteinase inhibitor aprotenin (Trasylol), which is avidly pinocytosed by the kidney, Just and Habermann [124] found that this protein is bound to the brush border with saturation kinetics. The binding apparently depends on the isoelectric point (IP) of the protein. For instance, the neutral tetramaleolyl derivative of aprotenin with IP '- 7.0 is not bound, while the basic guanidyl derivative (IP 10.5) is bound to the same extent as the genuine aprotenin. Other peptides like bradykinin, glucagon and insulin are also bound, but to a smaller extent and independently of the concentration added. It is suggested that binding is the first step of pinocytosis. Aprotenin, furthermore, is not degraded by the aminopeptidase which is located at the surface of the brush border [125], while other peptides, such as angiotensin II, seem to be split,

that way without having entered the kidney cells [136].

Ill

Fig. 9. Schematic diagram of cellular protein absorption in the proximal tubule, presented on the basis of observations on ferritinabsorbing tubular epithelia of the rat kidney, up to three hours after the start of absorption (simple vacua/es are characterized by thin membranes and lysosomal vacuoles are characterized by thick membranes). I. phase: Micropinocytosis at the base of the brush border up to apical absorption vacuoles. Mvb multivesicular body with

stored ferritin. Il. phase: a) Ferritin-filled vacuoles which have migrated in basal direction = "primary" protein absorption-droplets; b) pre-existing lysosome; b') fusion between a "primary" absorption droplet and a pre-existing lysosome; b") pre-existing lysosome with an incorporated protein absorption-droplet; c) dense protein droplet (comparable to a) with demonstration of acid phosphatase, probably a protein absorption-drop directly transformed into a lysosome. GZ = Golgi-zone with a few acid phosphatase positive cisternae (painted black). Ill, phase: Release through the basal membrane of low-molecular fragments of the absorbed protein which were formed inside the lysosomes shown by double arrows [137].

lumen ferritin containing invagination occurs on the bases of the microvilli from which microvesicles are pinched off. These flow together and form larger (0.5 to 1.0 t) reabsorption vacuoles. Up to this stage the vacuoles are called primary protein absorption droplets or phagosomes. Later they fuse with enzyme-

The uptake step of pinocytosis was also studied by biochemical analysis of the primary pinocytotic yesides. Using horseradish peroxidase as marker which is also avidly pinocytosed, Bode et al [126] were able to isolate primary pinocytotic vesicles. These vesicles

contain none of the typical brush border enzymes such as alkaline phosphatase, aminopeptidase and 5'-

nucleotidase [127]. Furthermore, they incorporate precursors of proteins ('4C-guanido-arginine), of glycoproteins (3l-1-fucose) and of phospholipids (3H-

myoinositol) faster than do the brush border membranes [128]. Both findings indicate that pinocytosis is

accompanied by a rapid de novo synthesis of membrane material, which was earlier suggested already

by Thoenes, Langer and Wiederholt [116] on the basis of their morphologic studies. One of the most important findings seems to be that the pincytotic vesicles contain twice as much acidic phospholipids (phosphatidyl inositol + phophatidyl serine) but only half as much sphingomyelin as the microvilli membranes [127]. These acidic phospholipids may provide

the binding sites for the basic proteins which are preferentially pinocytosed [129]. Since papain had none and sialidase only a small effect on aprotenin binding to the brush border [124], one may suggest

145

Organic solute transport

that the basic proteins are bound first to the acid phospholipids. Then one may ask whether the bound proteins jump from binding site to binding site until they are enriched in the pinched-off region or whether they remain bound to the same acidic phospholipid molecules and diffuse together within the liquid membrane toward the pinched-off region. The finding of different acidic phospholipid turnover in the brush

border and pinocytic membranes so far seems to favor the first possibility. Turnover studies of the pinocytosed material indicate that it is digested and that the split products, but not the intact molecules,

are released into the interstitium and blood capillaries, respectively [130, 131].

One very important effect should be mentioned

molecular weight and polypeptide content per enzyme unit in preparations from the outer medulla of rabbit kidney. Biochim Biophys Acta 356:53—67, 1974

Isolation of a phlorizin sensitive glucose binding protein from brush borders of rat kidney cortex, in Vth mt Biophys Congr, Copenhagen, 1975, p. 74

6. THOMAS L, TSCFIOEPE W, TRAPP M:

7. KINNE, R, MURER H, KINNE-SAFERAN E, THEES M, SACHS G:

Sugar transport by renal plasma membrane vesicles: Characterization of the systems in the brush border microvilli and basal lateral plasma membranes. I Membr Biol 21:375—395, 1975 8. BUSSE D, STEINMAIER G: Osmotically reactive plasma mem-

brane vesicles prepared from rabbit kidney tubules by mild hypotonic lysis. Biochim Biophys Acta 345:359—372, 1974 9. ULIRiCII KJ, RUMRICH G, KLOSS S: Specificity and sodium

dependence of the active sugar transport in the proximal convolution of the rat kidney. Pflflgers Arch 351:35—48, 1974

here, namely the carrier function of pinocytosed pro-

10. ULLRICH KJ, RUMRICH G, KLOSS 5: Sodium dependence of

teins. Gyory and Kinne [132] observed that antimycin A injected into the tubular lumen does not

II. LOESCHKE K, BAUMANN K: Kinetische Studien der -Gluco-

poison the kidney cells unless it is injected together with albumin. Since it was observed that antimycin A is readily bound to albumin [133], the simplest explanation for this finding is that antimycin A bound to

plasma albumin is taken up into the cell by pinocytosis. Within the cell the bound antimycin is liberated and diffuses out of the pinocytotic vacuole into the cytoplasm. A similar pin ocytic carrier function was observed by Eybl and Ryser [129, 134] for ferritin in the uptake of cadmium. A quantitative study of the pinocytosis of different macromolecules using micropuncture techniques has not been performed so far. Since by intraluminal application of a labelled macromolecular substance and subsequent rinsing with the same, but unlabelled, substance it should be possible to discriminate between the amount which was bound and that which was already taken up by the cell, a quantitative analysis of pinocytosis seems to be feasible. Reprint requests to Dr. K. J. Ullrich, Max-Planck-Institut Jir Biophysik, 6 Frankfurt/Main, Kennedyalle 70 West Germany.

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