Pfl/igers Arch (1992) 421:455-465

Journal of Physiology 9 Springer-Verlag 1992

Substrate specificity of the luminal Na +-dependent sulphate transport system in the proximal renal tubule as compared to the contraluminal sulphate exchange system C. David and K. J. Ullrich

Max-Planck-Institut f/ir Biophysik, Kennedyallee70, W-6000 Frankfurt am Main 70, Federal Republic of Germany ReceivedApril 17, 1992/AcceptedJune 3, 1992 Abstract. The efflux of [3SS]sulphate from the lumen of the proximal renal tubule into tubular cells of rats was measured by the stop-flow tubular-lumen microperfusion technique. The transport parameters obtained and the apparent Ki values of competing substrates were compared with those of the contraluminal influx of [35S]sulphate from the interstitium into tubular cells. For the luminal sulphate efflux a Kin(l, S O l - ) of 0.8 mmol/1 and a Jmax(1, SO~-) of 0.2 pmol s -1 cm -1 were found. The corresponding contraluminal values were Km(cl,SO 2-) 1.4 mmol/1 and Jmax(cl, SO 2-) 1.2 pmol s - 1 cm- 1. Omission of Na + from the perfusates reduced the luminal efflux of sulphate by 8 3 %, while the contraluminal influx of sulphate was not changed. Increase in HCOg concentration inhibited both luminal efflux and contraluminal influx of sulphate, while a change of pH from 6.0 to 8.0 was without effect. Comparing the apparent Ki(SO 2-) values for luminal and contraluminal sulphate transport, a relationship close to 1:1 was seen for some inorganic substrates with tetrahedral molecular structure (thiosulphate, sulphate, molybdate and selenate). The same holds for phosphate, while for oxalate the contraluminal Ki(SO 2-) value was lower than the luminal one (1.2 and 4.5 mmol/1). Some of the dicarboxylates and disulphonates tested show the same affinity to the luminal Na +dependent sulphate transporter and the contraluminal sulphate exchange system, whereas most of the benzene carboxylate and benzenesulphonate derivatives tested exhibit higher luminal than contraluminal Ki values. The inhibitory potency increased with rising numbers of substituents on the benzene ring. This effect was more pronounced for the contraluminal sulphate transporter. In general, only disulphonates and analogues as well as similarly structured compounds (5-sulphosalicylate, 2hydroxy-5-nitrobenzenesulphonate, eosine-5-isothiocyanate) have a good inhibitory potency toward the luminal sulphate transporter [apparent Ki 0.9-3.1 mmol/1]. All the tested sulphamoyl and phenoxy diuretics, and fluorescein and phenolphthalein dyes showed no or a smaller inhibitory potency to the luminal sulphate transport sysCorrespondence to: K. J. Ullrich

tern than to the contraluminal. The most effective inhibitors of both sulphate transport systems are 8-anilino-1naphthalenesulphonate, orange G, and H2-DIDS. The data indicate that the Na+-dependent luminal and the Na+-independent contraluminal sulphate transport systems accommodate a similar spectrum of anionic substrates, whereby the inhibitory potency against the luminal Na+-dependent sulphate transport system is identical or smaller than against the contraluminal transporter. Key words: Oxalate - Benzenecarboxylates - Benzenesulphonates and salicylates - Sulphamoyl diuretics Fluorescein and phenolphthalein dyes

Introduction

In the mammalian kidney sulphate is filtered in the glomeruli and reabsorbed by the renal tubules to a large extent [4, 16]. Stop-flow and micropuncture experiments revealed that sulphate reabsorption occurs in the proximal tubules [8, 13]. The Na + dependence of renal sulphate reabsorption was demonstrated in micropuncture experiments [26] and experiments with isolated brushborder membrane vesicles [15, 20]. It was thus found that reabsorption of sulphate in cotransport with Na + is electroneutral [6, 15, 18], saturable [15] and inhibited by thiosulphate and molybdate [2, 6, 15, 18, 26]. The tetrahedral structure of these molecules was considered a main criterion for their interaction with the luminal sulphate transport system. Transtubular Na +-dependent sulphate transport is not influenced by pH changes [15, 26] but it depends on the presence of bicarbonate [26]. Similarly Na+-dependent sulphate uptake into brushborder membrane vesicles did not show significant pH sensitivity [15], but bicarbonate dependence was not tested. At the basolateral cell membrane, sulphate transport is Na +-independent and mediated by a transport system

456 that works by exchanging sulphate with thiosulphate, oxalate, bicarbonate, h y d r o x y l ions, and a variety o f organic anions [2, 12, 14, 17", 22]. Experiments on the rat kidney in situ revealed that contraluminal sulphate/ sulphate exchange depends on the presence o f bicarbonate and chloride [30] while bicarbonate trans-stimulation o f sulphate u p t a k e in basolateral vesicles indicated that bicarbonate exchanges directly with sulphate and n o t via a t r a n s m e m b r a n e p H difference [14]. In situ u p t a k e experiments showed, for the contraluminal sulphate exchanger, a b r o a d specificity for organic anions, e.g. sulphonates, carboxylates, sulphocarboxylates and their derivatives, salicylate analogues, sulpho dyes and sulphamoyl compounds [30-34]. 4 - A c e t a m i d o - 4' - isothiocyanatostilbene - 2,2" - disul phonic acid (SITS) and 4,4'-diisothiocyanatostilbene2,2'-disulphonic acid (DIDS), inhibitors o f the red cell anion exchanger, inhibited luminal N a § c o t r a n s p o r t weakly [1, 2] or n o t at all [6, 26], while they exhibited a high affinity to the contraluminal sulphate transporter [1, 14, 22, 32]. The intention o f the present study was to examine whether and to w h a t extent overlapping substrate specificities exist between the luminal, N a + - d e p e n d e n t sulphate transport system and the contraluminal sulphate/ anion exchanger. We demonstrate that the luminal N a § dependent sulphate transport system interacts also with oxalate and bicarbonate and that it displays for tetrahedral substrates as well as for some dicarboxylates and disulphonates the same affinity as the contraluminal sulphate/anion-exchange system. F u r t h e r m o r e it was f o u n d that salicylate and related c o m p o u n d s inhibit the luminal N a + - d e p e n d e n t sulphate transport system with a lower p o t e n c y as c o m p a r e d to the contraluminal sulphate/anion-exchange system.

Materials

and methods

The experiments were performed in male Wistar rats (Winkelmann, Kirchborchen, FRG) with a body weight of 180-200 g and fed on Altromin standard diet and with free access to tap water. They were anaesthetized by injection of Inaetin (Byk Gulden, Konstanz, FRG) 120-150 mg/kg body weight intraperitoneally and placed on a heated operating table (thermostat control 37~C). An incision was made on the left flaI~k and the kidney was separated from the surrounding fascia. The capsule was stripped off and the kidney immobilized in a plastic cup, resting on cotton wool and covered by paraffin oil heated to 37~ Using the stop-flow tubular microperfusion method as described in previous work from this laboratory [21] the tubular lumen was punctured with three different sharpened glass pipettes (tip diameter 5 - 8/am): one containing blue-coloured castor oil to block the flow of ultrafiltrate in the proximal tubule, a second one very nearby containing steady-state solution with 35S-labelled sulphate (0.01 mmol/1) splitting the oil blockade column when injected, and a third one at a distinct distance, but in the same tubule, for collecting the steady-state solution after 2 s contact time (acoustic signal). The steady-state solution also contained 14C-Iabelled inulin as a volume marker. The reaspirated sample with a volume of 1 * In the proximal tubule of the flounder, where sulfate is net secreted, compared to net reabsorption in mammals, the luminal sulfate transporter is a Na +-independent exchanger

2 nl was diluted in 10 lal Ringer solution and pipetted directly into 5 ml Pico-Fluor 15 (Packard, Frankfurt, FRG) scintillation fluid. The [3SSJsulphate and [14C]inulin radioactivity was measured in a Nuclear Chicago liquid scintillation counter. The steady-state solution for luminal perfusion contained (in mmol/t): Na + 134.0, K + 4.0, Ca 2+ 1.5, Mg 2+ 1.0, C1- 136.5, HCO~- 4.0, raffinose 31.0 (gassed with 95% 02, 5% CO2) [21] and [35S]sulphate 0.01. Under Na+-free conditions it contained K + 4.0, Ca 2+ 1.5, Mg z+ 1.0, CI145.0, D-glucamine 138.5 and raffinose 15.5 (gassed with Oz), and under Cl--free conditions: Na + 142.0, K + 4.0, Ca 2 + 1.5, Mg 2 + 1.0, gluconate 148.5 and raffinose 15.5 (gassed with 02). The solution for capillary perfusion contained (in mmol/1) control: Na + 150.0, K + 4.0, Ca 2+ 1.5, Mg 2+ 1.0, C1- 131.5 and HCOj 25 (gassed with 95% 02, 5% CO2); under Na+-free conditions: K + 4.0, Ca z+ 1.5, Mg 2+ 1.0, CI- 156.5 and D-glucamine 150.0 mmol/1 (gassed with Oz); under C1--free conditions: Na + 150.0, K + 4.0, Ca 2+ 1.5, Mg 2+ 1.0 and gluconate 156.5 mmol/t (gassed with 02). All added substances replaced an equivalent amount of Na + or CI- to maintain constant osmolarity. Further specifications are given in the legends to the tables and figures. The conditions for evaluation of the contraluminal transport parameters are described in [34]. The source and specific activities of the radioisotopes used are as follows: [35S]sulphate with a specific activity of 1.4 Ci/mmol, and [lgc]inulin with a specific activity of 15 mCi/g were purchased from DuPont NEN, Dreieich, FRG. The sources of the inhibitors used are indicated in Table 1.

Calculation of kinetic data." (Figs. 1 and 2) By using the stop-flow tubular microperfusion technique, solutions with different concentrations o f sulphate were injected into the proximal tubules and reaspirated after different c o n t a c t times9 Figure 1 shows the time-dependent loss o f sulphate f r o m the tubular lumen at starting concentrations o f 0.01 - 10.0 retool/1. In order to investigate p r e d o m i n a n t l y the luminal transport step, a contact time o f 2 s was chosen since luminal c o n c e n t r a t i o n changes were p r o p o r t i o n a l to time and o f appropriate m a g n i t u d e during this period (Fig. 1). Flux rates related to tubular length were calculated as J = V Ac 1- 1 s - 1, where V is the v o l u m e o f the injected fluid c o l u m n and 1 the length o f the fluid c o l u m n and V I - I = r2rc, where r = 15 g m is the mean radius o f the tubular lumen [27]. A n Eadie-Hofstee plot o f the efflux values at different starting concentrations (Fig. 2) gives a straight line with a Km(1, S O ] - ) value o f 0.8 mmol/1 and a Jmax(1, 8024 - ) value o f 0.2 p m o l c m - 1 S-1. Since there is apparently no significant diffusional c o m p o n e n t involved in sulphate efflux f r o m the tubular lumen, apparent Ki values can be evaluated by: Ki, app. =

ACi Ac e - A c i

K m [I] [S]4-Km'

where K m = app. Km(l, SO42-) o f the test substance (0.8 mmol/1), [S] = c o n c e n t r a t i o n o f the test substance (substrate 0.01 mmol/1) and [I] = c o n c e n t r a t i o n o f the inhibitor. Aco and Ac~ refer to the substrate concentration change under control conditions and in the presence o f the inhibitor respectively. Since the chosen substrate c o n c e n t r a t i o n o f 0.01 mmol/l is small in relation to Km (0.8 mmol/l), the second term can be a p p r o x i m a t e d by [I]. The calculated app. K~ values are used as operational values for c o m p a r i n g the a p p a r e n t inhibitory potencies

457

of the various test substances. Competitive inhibition is assumed but not explicitly tested. It should be mentioned that even the hydrophobic fluorescent probe 8-anilinol-naphthalene-sulphonate shows competitive inhibition, i.e. nearly the same Ki values [Ki(1, S O 4 2 - ) = 0 . 2 0.3 mmol/1] at different applied substrate concentrations (0.1-1.0mmol/1). The apparent Ki values for the contraluminal sulphate transport system are taken from previous publications from this laboratory [30-34]. All data presented are mean values ( + SE).

Results

Effect of Na +, CI-, HCO~ and pH on luminal sulphate efJTux: (Fig. 3) Omission of Na § from the perfusate (Fig. 3) reduces the efflux of [3SS]sulphate through the luminal brush-border membrane by 83%. Omission of chloride from the perfusate and compensation with gluconate, which does

not interfere with the sulphate transporter [14, 30], shows no effect on the luminal sulphate efflux while an increase of HCO~ concentration from 4 mmol/1 (gassed with 5% CO2; pH 6.6) to 50 mmol/1 (gassed with 10% COz; pH 7.4) causes a 21% inhibition of the luminal sulphate efflux. Changes between pH 6 and 8 of HEPES-buffered luminal perfusates did not change sulphate efflux.

Comparison of@parent Ki( SO 2-) values of tetrahedral molecules for the luminal and contraluminal sulphate transporters: (Table 1 and Fig. 4) In Fig. 4 the apparent luminal Ki(SO]-) values of some inorganic tetrahedral molecules, thiosulphate, sulphate, molybdate and selenate, as well as phosphate and oxalate, are plotted against the respective contraluminal values.

0.20 Ct

[% 100~----~~--I ~\\

N

....................... ~

i tl;t]n-g-c. . . . .

~ " - ' - - ~

trotion

[mm0t/t]

'T E o

-5 E

ou')

0

ii

Km

=

0.80 m m o l / I

Jmax

=

0.21pmol'cm-l's

-!

0.15

0.10

0.0 5

0,3

o.ol 0

,~ Time

[s]

Fig. 1. Kinetics of sulphate efflux from the proximal tubule of the rat kidney into renal cortical tubular cells. Decrease in luminal sulphate concentration (q) with contact time at different starting concentrations. The figure shows means + SD of 1 0 - 3 0 measurements from three to ten rats

100%

0

o

o So

0.20

o;o

[sol-] Fig. 2. Eadie-Hofstee plot of the 2 s sulphate efflux values, related to tubular length, for different sulphate starting concentrations. Further details are as stated in the legend to Fig. l

= 0.0t0

--I-0,008 0.006

-

--I--***

Fig. 3. Effect of Na +, C1- and HCO;-, as well as changes in pH, in the luminal perfusate on 2-s luminal ---T. . . . . ~js_. -In - - z - NS- Z z - - - - - - f Z S efflux of sulphate into renal cortical tubular cells of 0,002rats. Simultaneously the peritubular capillaries were perfused with solutions in which the respective ions Owere also replaced. Starting concentration 0.0t nmol/1 [mmot/[l sulphate. - - F - , Mean + SE. Each value represents CI,2s 7 - 1 2 measurements from three to four rats. Further control contro[ controt c0ntro[ NQ§ Na+-free C[C[--free HCO] pH6.0 pH6.B pH8.3 details are as stated in the legend to Table 1. P was (C[,Os-%2s ] (130mmoL/[) (G[ucamine) {133mmot/t)(G[uconote) (GHCO~ mmoUl)(50mmo[d) calculated from an unpaired t-test: * P < 0.05; (5~ (10~ HE PES-buffer{I0 mrno{/[ 9 * P < 0.01 ; *** P < 0.001 0.004

-

-

-

- - T - -

.

.

.

.

.

.

.

.

.

.

.

.

.

.

458 Table 1. Effect of tetrahedral and other divalent anions, carboxylates, sulphonates, sulphocarboxylates and derivatives, dyes and diuretics on the 2-s/4-s decrease &luminal and contraluminal sulphate concentration (control Ar luminal = 70.8 -+ 0.8%) a Substances added u

Fig. 4 Thiosulphate (m) Motybdate (m) Selenate (m) Sulphate (m)

Phosphate (a) Oxalate Fig. 5 Benzoate (a) 2-Hydroxybenzoate (salicylate) (a) 2-Hydroxy-5-chlorobenzoate (5-Chlorosalicylate) (e) 2-Hydroxy-3,5-dichlorobenz0ate (hoe) 1,4-Dihydroxy-2,5-dichlorobenzene (k) 2-Hydroxy-5-nitrobenzoate (hoe) 2-Hydroxy-3,5-dinitrobenzoate (e) 2-Hydroxy-3,5-dinitrobenzaldehyde (a) 1,3-Benzenedicarboxylate (e) 2-Hydroxy-l,5-benzenedicarboxylate 3-Carboxy- 1-benzenesulphonate (k) 3-carboxy-4-hydroxy-benzenesulphonate = 2-hydroxy5-sulphobenzoate = 5-sulphosalicylate (m) 3-Carboxy-4-ethylamino-l-benzenesulphonate(hoe) Benzenesulphonate (f) 2-Hydroxy-benzenesulphonate (hoe) 2-Hydroxy-5-nitrobenzenesulphonate (hoe) 1,3-Benzenedisulphonate (r) 4-Hydroxy- 1,3-benzenedisulphonate (hoe) 4,6-Dihydroxy-l,3-benzenedisulphonate (hoe)

(mmol/1)

Ae

app. Ki _+ SE (mmol/l)

(% + sg) luminal

luminal (1)

contraluminal (cl)

0.1 1.0 5.0 3.0 1.0 0.3 0.01 50.0 5.0

42.8 _+ 3.4 42.2 _+ 3.5 17.9 _+ 3.0 15.3 _+ 2.6 32.4 _+ 2.0 54.7 _+ 2.8 70.8 __.%0.8 29.5 -+ 2.3 34.9 + 2.0

0.2 _+0.09 1.5 _+0.6 1.6 _+0.5 K m = 0.80

0.3 _+ 0.07 0.8 _+0.3 1.4 _+ 0.8 Km = 1.4

0.7 2.0 1.2 0.6

44.6 _+48.7 4.5 _+ 0.7

32.0 _+ 10.0 1.2 _+0.6

1.4 3.7

20.0 20.0

48.8 -+ 1.8 61.5 _+2.7

36.1 _+ 10.9 NS ( > 75)

NS (> 25) NS ( > 40)

5.0 5.0 2.0 5.0 5.0 5.0 20.0 5.0 20.0

73.5 _+ 1.5 50.9 _+ 1.5 56.2 • 2.9 66.7_-/-3.5 54.7 -+ 2.8 51.5 -+ 3.9 17.7 _+ 1.7 45.0_+2.9 38.0 • 2.5

NS ( > 37) 12.5 _+2.2 10.0 -+ 5.3 NS (> 37) 13.7 -+ 5.4 13.6 _+ 5.5 6.5 _+ 1.1 11.1 _+3.2 22.2 _+ 4.8

3.4 _+ 2.5 0.4 _+0.1 0.2 _+ 0.05 4.2-+3.7 0.7 ___0.2 3.6 _+2.8 2.7 _ 1.6 0.8_+0.2 NS (> 25)

1.0 20.0 20.0 5.0 5.0 5.0 5.0 5.0

36.5 _+ 2.5 39.1 _+4.2 48.2 _+ 3.2 54.3 + 2.1 17.3-+3.3 24.5 -+ 3.1 36.9 -+ 2.4 38.6_+2.4

0.9 _+ 1.1 22.7 _ 6.4 47.0_+ 16.4 25.1 -+ 11.9 1.5-+0.4 2.6 -+ 0.7 5.7 _+0.8 6.4_+1.8

0.8 _+ 0.7 0.7 _+0.1 NS (> 25) NS ( > 14) 0.6_+0.2 6.1 -+ 6.9 3.0 -+ 2.0 NS (> 25)

Fig. 6 Benzoate (a) 2-Hydroxybenzoate (salieylate) (a) 2,4-Dihydroxybenzoate (e) 2,6-Dihydroxybenzoate (e) 2,3-Dihydroxybenzoate (e) 2,5-Dihydroxybenzoate (e) 1,3-Benzenedicarboxylate (e) 1,3,5-Benzenetricarboxylate (e) 1,2,3-Benzenetricarboxylate (e) Pyridine-2,4-dicarboxylate (e) 3,4-Carboxy-l-benzenesutphonate (e)

20.0 20.0 20.0 20.0 20.0 20.0 20.0 10.0 10.0 20.0 5.0

48.8 -+ 1.8 61.5 -+ 2.7 62.4_+ 4.0 60.8 _+ 3.2 61.5 + 5.0 66.1 -+ 3.9 17.7 -+ 1.7 65.4 _+ 3.4 40.8_+3.2 21:3_+0.4 35.9 -+ 2.4

36.1 _+ 10.9 NS (> 75) 73.4_+ 29.8 65.4 _+ 21.0 NS (> 75) NS (> 75) 6.5 -+ 1.1 NS (> 75) 12.9_+2.9 8.1_+0.4 6.6 -+ 1.7

NS (> 25) NS (> 40) 4.5 _+ 4.0 2.3 _+ 1.3 NS ( > 23) NS (> 17) 2.7 -+ 1.6 1.5 _+ 0.6 0.9_+0.3 5.4_+6.3 1.9 -+ 0.9

Fig. 7a 3-Sulphamoyl-4-phenoxybenzoate (hoe) Furosemide (hoe) Piretanide (hoe) Hydrochtorothiazide (s) Tienilic acid (hoe) Ethacrynic acid (msd) Probenecid (s)

5.0 20.0 20.0 5.0 10.0 20.0 20.0

81.8 _+4.2 44.5 -+ 3.3 49.5 -+ 3.3 81.1 _+2.7 69.5 _+4.6 25.6-+1.6 39.1 -+ 2.8

NS (> 37) 36.0 + 13.5 46.5 _+ 14.5 NS ( > 37) NS (> 75) 11.3-+1.6 23.6 + 5.0

0.8 _+0.2 0.9 + 0.3 2.9_+ 1.9 4.4_+4.3 2.7 + 1.6 4.7-+4.8 7.9 -+ 2.2

51.6_+2.5 26.9_+3.0 75.2 -+ 2.7 56.5 _+ 3.8

13.2--+3.8 3.1_+0.8 NS (> 37) 27.7 + 16.5

0.2_+0.03 0.2_+0.01 0.7 + 0.2 5.3 _+ 5.9

Fig. 7b Eosine Y (a) Eosine-5-isothiocyanate (s) Fluorescein (m) Sulphofluorescein c

Ratio: app. K~, 1/ app. Ki, cl

5.0 5.0 5.0 5.0

30.6 41.5 20.1 3.8 2.4 13.5 1.1 25.2 2.3 0.4 1.9

16.3 28.4 2.4 15.0 1.5 3.5

41.4 16.0 2.4 3.0

69.5 15.5 5.2

459 Table 1 (continued) Substances added b

Bromphenol blue (m) Phenol red (s) Bromcresol purple (m) Fig. 8 8-Anitino-l-naphthalenesulphonate (f) (ANS) Orange G (f) H2-DIDS (s)

Ac

app. Ki+_SE (mmol/1)

(% • SE) luminal

luminal (1)

contraluminal (cl)

5.0 5.0 20.0

35.6 • 2.3 74.8 • 3.7 39.3 • 2.8

5.1 • 0.8 NS (> 37) 22.0 • 5.2

0.1 + 0.03 1.7 • 0.8 2.5 • 1.3

46.4

1.0 0.3 0.1 1.0 5.0

13.8 _+2.5 28.9 ___2.6 45.7 4- 2.4 29.9 _ 1.0 15.3 • 1.8

0.3 • 0.2 • 0.2 + 0.7 • 1.3 •

0A • 0.02

3.0

0A • 0.03 0.4 + 0.1

6.6 3.3

(mmol/1)

0.07 0.04 0.04 0.1 0.2

Ratio: app. Ki, 1/ app. Ki, cl

8.8

a Ac is the decrease in luminal [3SS]sulphate concentration within 2 s as a percentage 4- SE of the starting concentration of 0.01 mmol/1. The apparent Ki(SO2-) values luminal were evaluated and contraluminal taken from [30-34]. NS, no significant inhibition was observed compared with the controls; > x, an apparent Ki value, if present at all, must be larger than x mmol/1. For the calculation of the apparent Ki values the decrease of the concentration of the inhibitory substance was not taken into account. b Source of inhibitors used: (a) Aldrich-Chemie, Steinheim, FRG; (e) EGA-Chemie, Steinheim, FRG; (f) Fluka GmbH, Neu-U1m, FRG; (hoe) Dr. H.-J. Lang, Pharmasynthese, Hoechst AG, Frankfurt/Main, FRG; (k) Kodak, Rochester, N.Y., USA; (m) Merck, Darmstadt, FRG; (msd) Merck, Sharp & Dohme, Miinchen, FRG; (r) Riedel-de Hahn, Seelze, FRG; (s) Sigma, Deisenhofen, FRG; (v) Serva, Heidelberg, FRG c Sulphofluorescein was kindley provided by Prof. Dr. M. Steinhausen, Heidelberg, FRG

A relation close to 1:1 is obtained, i.e. these anions show approximately equal inhibitory potencies against the sodium-dependent luminal and the sodium-independent contraluminal sulphate transport system. An exception is oxalate, for which the inhibitory potency to the luminal sulphate transporter is lower [Ki(1, SO 2 - ) = 4.5; Ki(cl, SO 2 - ) = 1.2 mmol/ll.

100-

E -6 ;-

Effect of benzenecarboxylate and benzenesulphonate derivatives on luminal and contraluminal sulphate transport: (Table 1 and Fig. 5) Figure 5 shows the chemical formulas of some carboxylate and sulphonate derivatives and their apparent Ka values against luminal sulphate transport; the respective contraluminal values are given in brackets. Substances depicted within the framed area have comparable Ki values for luminal and contraluminal sulphate transport; i.e. the Ki values deviate by a factor of maximally 2.5. Substances plotted outside the framed area show at least 15-fold differences in their Ki values whereby the luminal Ki value is mostly higher than the respective contraluminal one. (The following description of results is guided by the orientation arrows.) Starting f r o m benzoate and benzenesulphonate and their 2-hydroxy derivatives, which show no or only very small inhibitory potencies to both sulphate transport systems, we tested 1,3-benzene dicarboxylate and 1,3benzenedisulphonate [Ki(1, SO 1 - ) = 6.5 and 2.6 m m o l / 1; Ki(cl, SO 2 - ) = 2.7 and 6.1 mmol/1 respectively]. A combination with both substituents, 3-carboxy-l-benzenesulphonate, interacted luminally very weakly [Ki(1,

~o-

1.0,./

0.1 0.1

I

I

I

1.0

10

100

Ki,so]-vatues contraluminal [mmot/t] Fig. 4. Plot of apparent (app.) Ki(SO2-) luminal values against app. Ki(SO2-) contraluminal values for some inorganic tetrahedral molecules and oxalate. Further details are as stated in Table 1

SO ] - ) = 22.2 mmol/1] and contraluminally not at all. Introduction of an O H group in position 2 in this molecule, yielding 5-sulphosalicylate, resulted in high inhibitory potencies to both sulphate transport systems [Ki(1, SO 2 - ) = 0.9 mmol/1 and Ki(cl, SO 2 - ) = 0.8 mmol/1]. Interestingly a similarly high inhibitory potency to that with 5-sulphosalicylate is seen with 2-hydroxy-5-nitrosulphonate [Ki(1, SO42-) = 1.5 mmol/1 and Ki(cl,SO 2 - ) = 0.6 mmol/1 respectively]. A second O H group in position 4 (2,4-dihydroxy-l,5-benzenedisulphonate) does not

460 0 ~~.lHO H

13.6(3.6)

02N

" ~OH Cl 10.0 (0.2)

NO2

,~ , K i ,S024" 'l

CI

[Ki'cl'S04) OH

c0o,

02N

oo.

NO2

CI

NS>37 ( 4.2 )

~' COOH .j~OH 02N

C[ 600H

NS > 37

COOH

11 1 "

COOH

.j~OH(3'4) ~foH(O'8) Cl

HOOC"v

~NHC2H5

22,7

(o.7)

H03S- v

NS > 75 COOH (NS > 40) ~J-~r.OH

36,1

COOH

(NS>25) 0

.

COON 6,5

.f~

"

HOOC ~"~.~,~,,. 47.0

~

COOH 222 COOH 0.9 (0.8) HO3S.J~ (NS>25)-- J ~ OH H03S

mmol/1 and 12.5 retool/1 respectively. However, all four compounds possess a good inhibitory potency towards the contraluminal sulphate transporter with K~(cl, SO~-) values of 4.2, 3.4 and 0.7, 0.4 mmol/1 respectively. Furthermore, 1,4-dihydroxy-2,5-dichlorobenzene shows the same inhibitory potency as 2-hydroxy-3,5-dichlorobenzoate with Ki(1, SO4a-) values of 10.0 and 12.5 retool/1 and contraluminal values of 0.2 and 0.4 mmol/1. 2-Hydroxy-3,5-dinitrobenzaldehyde showed similar luminal inhibitory potency, and a somewhat smaller contraluminal inhibition compared with that shown by 2-hydroxy-3,5-dinitrobenzoate. These data indicate that with increasing charge accumulation on the benzene ring the inhibitory potency to both luminal and contraluminal sulphate transport systems increases, an effect usually seen with the contraluminal system more than with the luminal one. The spatial charge distribution seems to be an important factor to explain the differences in luminal and contraluminal inhibitory potencies.

(6.1) H%S 25.1

,S03H

(m>(o.6)16

14) ~"~176 ~HrS%H

$03H

5.7

H%s~ O H (30)

02Nr'~v...-S03H

S~ ' ~ O H 6.4 H03 - O~H (NS>25)

Fig. 5. Effectof benzenecarboxylateand benzenesulphonate derivatives on 2-s luminal efflux (4-s contraluminal influx) of sulphate into renal cortical tubular cells. Starting concentration 0.01 mmol/1 sulphate. The decrease of this concentration within 2 s (4 s) contact time ACl,2s was 7.08 Ilmol/1 (Ace1,4s = 4.7 gmol/1) under control conditions. Each value represents the mean of 6-11 samples from two or three animals (7-8 samples from two or three animals). The arrowsindicate a change in one sidegroup, not a metabolicpathway. Further details are as stated in Table 1

change the luminal interaction but abolishes the contraluminal inhibitory potency. This is the only disulphonate tested where a lower luminal than contraluminal Ki(SO42-) value was observed. Introduction of an OH group in position 2 in 1,3-benzenedicarboxylate resulted in a very low Ki(cl, S O l - ) value (0.8 retool/l) but had the opposite effect on the luminal interaction with the sulphate transporter [Ki(1, S O l - ) --- 11.1 retool/l]. The same tendency was seen with the introduction of one NHCzH5 group in position 2 in 3-carboxy-l-benzenesulphonate [Ki(cl, SO]-) = 0.7 retool/l; Ki(1, SO 2-) = 22.7 retool/l]. With 2-hydroxybenzoate (salicylate) and analogues the effect on sulphate transport of introducing one or two NO2 or C1 groups was investigated. 2-Hydroxy-5-nitrobenzoate and 2-hydroxy-5-chlorobenzoate still do not interact with the luminal sulphate transport system. They need a second electronegative group, as in 2-hydroxy-3,5-dinitrobenzoate and 2-hydroxy-3,5dichlorobenzoate with luminal Ki(1, SO 2-) values of 13.7

Effect of benzenedihydroxy and benzenepolycarboxy derivatives on luminal and contraluminal sulphate transport: (Table 1 and Fig. 6) As demonstrated in Fig. 6 benzoate shows, as already mentioned, almost no inhibitory potency with both sulphate transport systems. With 1,3-benzenedicarboxylate a weak interaction was observed [Ki(1, SO 2-) = 6.5 and Ki(cl, S O l - ) = 2.7 retool/l]. Addition of a third COOH group produces different results, depending on the position in the molecule. 1,3,5-Benzenetricarboxylate did not inhibit the luminal sulphate transport system but 1,2,3-benzenetricarboxylate showed a Ki(1, SO 2-) value of 12.9 retool/1. The contraluminal Ki values ranged from 1.5 retool/1 to 0.9 retool/1. 2-Hydroxybenzoate (salicylate) did not interact with sulphate transport. Introduction of a second OH group in distinct positions of the molecule caused different effects: 2,3- and 2,5-dihydroxybenzoate both showed no inhibitory potency while 2,4- and 2,6-dihydroxybenzoate inhibited the luminal sulphate transport weakly [Ki(1, S O l - ) values of 73.4 and 65.4 retool/l], but interacted well with the contraluminal sulphate transporter [Ki(cl, SO42-) values of 4.5 and 2.3 retool/l]. These data indicate again that charge accumulation and distribution determine the interaction with the sulphate transporters and are responsible for different affinities towards the luminal and the contraluminal sulphate transport system. Effect of some sulphamoyl and phenoxy diuretics as well as fluorescein and phenolphthalein dyes on luminal and contraluminal sulphate transport: (Table 1 and Fig. 7a and 7b) As shown in Table 1 and Fig. 7 a all tested sulphamoyl diuretics: 3-sulphamoyl-4-phenoxybenzoate, furosemide, piretanide, hydrochlorothiazide and also probenecid, as well as the tested phenoxy diuretics: tienilic acid and

461 COOH 0H

NS > 75

~

(NS > 17)

COOH ~,,,.H O - ~ O H

65.4 ( 2.3 )

Ki,I ,S024

H0/~_. -

NS > 75

COOH

(Ki,cl,S024 ) I

COOH ,~OH

(NS > 23)

73.4 ( 4.5 )

/ o. COOH NS > 75 (NS > 40) G O H

36.1 (NS > 25)

COOH NS > 75

COOH 6.5

~COOH

(~

(2.7)

v-COOH

(1.6) =

HOOC

COOH

COOH 8.1 (5.4)

HOOC COOH

,~COOH H03S

6.6 (1.9)

ethacrynic acid, exerted a good to moderate inhibitory potency against contraluminal sulphate transport while they interacted with the luminal sulphate transport rather weakly, if at all. The same holds for fluorescein and phenolphthalein dyes (Table 1 and Fig. 7b). These dyes exert a high to moderate inhibitory potency toward the contraluminal sulphate transporter. Only two of them, i.e. eosine-5-isothiocyanate and bromphenol blue, exhibited a moderate inhibitory potency toward the luminal sulphate transporter [Ki(1,SO~-) = 3.1 and 5.1 mmol/1 respectively].

Substances with high inhibitory potency for luminal and contraluminal sulphate systems." (Table 1 and Fig. 8) Figure 8 shows the chemical formulas o f some substances with the highest inhibitory potencies for both the luminal and the contraluminal sulphate transport systems: 8anilino-l-naphthalenesulphonate with KI(1,SO42-) = 0.2 mmol/1 and Ki(cl, SO 2-) = 0.1 mmol/1, and the disulphonates orange G with Kid, S O l - ) = 0.7 retool/1 and Ki(cl, SO 2-) = 0.1 mmol/1, and Hz-DIDS with Kid, SO 2 ) = 1.3 retool/1 and Ki(cl, SO 2-) = 0.4 mmol/1.

COOH

[~COOH COOH

12.9

(O.9)

Fig. 6. Effect of benzenedihydroxy and benzenepoiycarboxy derivatives on 2-s luminal efflux (4-s contraluminal influx) of sulphate into renal cortical tubular cells. Starting concentration 0.01 mmol/1sulphate. The decrease of this concentration within 2 s (4 s) contact time Aq, 2s was 7.08 gmol/1 (Ace1,4s = 4.7 gmol/l) under control conditions. Each value represents the mean of 6 11 samples from two or three animals (7- 8 samples from two or three animals). The arrows indicate a change in one side group, not a metabolic pathway. Further details are as stated in Table I

The observed Na § dependence of the luminal sulphate transport system (Fig. 3) is in agreement with earlier data, obtained in micropuncture experiments [26] as well in experiments with isolated brush-border membrane vesicles [15, 20] and is in accordance with the notion that Na +-sulphate cotransport is electroneutral [6, 15, 18] and saturable [15]. In former studies in this laboratory a stimulatory effect of H C O 3 on transtubular sulphate reabsorption was observed [26]. Results of studies on basolateral membrane vesicles, where a trans-stimulation of sulphate transport by HCO;- was seen [7, 12, 14, 17"], allowed this effect to be explained by HCO~--driven sulphate exchange at the contraluminal cell side. Our present experiments show (Fig. 3) that H C O j also interferes with the luminal Na § dependent sulphate transport. Since the lumen-to-cell H C O 3 gradient is inwardly directed at the beginning of the proximal tubules and probably outwardly directed in later portions, it remains open whether the HCO~ concentration within the proximal tubular fluid has any significant effect on the luminal Na+-sulphate cotransport. Thus the straightforward mode for transtubular reabsorption of sulphate - and related substrates - seems to be electroneutral Na§ sulphate cotransport at the luminal cell side and exchange of sulphate against bicarbonate at the contraluminal cell side.

Discussion

Kinetics, Na +, and HCO~ dependence

Interference with oxalate

The apparent Km(1, SO 2 ) of 0.8 mmol/1 for sulphate transport from the tubular lumen into proximal tubular cells in situ (Fig. 2) is in good agreement with a Km of 0.6 mmol/1 for rabbit [20] and of 1.0 mmol/1 for rat renal brush-border membrane vesicles [23]. Furthermore, a missing diffusional term indicates that paracellular diffusion of sulphate is negligible.

Studies on the intact proximal tubule [26] as well as with basolateral membrane vesicles [12, 14, 17"] have shown that oxalate cis-inhibits and trans-stimulates contraluminal sulphate/anion exchange. Since, in all species tested so far, renal net secretion of oxalate has been observed [5, 11] it was hypothesized that the contraluminal uptake of oxalate would most likely occur in

462 3-Su[famoyt4-phenoxy-

a

~0 H2NOzS'~COO H

benzoate NS>37 (0.B)

Furosemide _ •36.0

C[~ H2NO2S

NH-CH2 COOH

Piretanide 46,5 (2.9)

H2NO2S'vCOOH

H (N A Cl HN.s"~SO2NH2

Ethacrynic acid 0--CHzCOOH 1 1.3 c[ cl (/,.7)

HsC2~c-COH2c/

Probenecid (CH3CHzCH2)2NSO2~COO H k~

Hydrochtorothiazide NS ~-37

o/~o

Br

NS>75 (2.7)

(0.9)

(~0

Br ' ~ ' * ~ " ~

Cco

Br H

Br Br HO.~O Br Br .~COOH SCN ~

HO ~COO

~H

O

0

~c ~ -so3H

"[ 3.2 (0,2)

(Ki,cl,SO

Br Br HO.~]. /~,o B r ' V [ ~ 5o3Br

HOL~ 1 ~.]~0 Eosine-5-isothiocyanat 3.1 {0,2)

Ftuorescein NS>37 (0.7)

SuEpho-

2 3.6

(7.9)

2Ki,l.,SO 4

(~.~)

Br

v -c 47.0 _ 73.4 • 65.4 • NS (> NS (>

NS (> 25) NS (> 40) NS (> 25) 4.5 • 4.0 2.3 • 1.3 NS (> 23) NS (> 17)

2.8 4.7 NS (> 20) NS (> 20) 19.1 9.3 9.5

10.9 75) 16.4 29.8 21.0 75) 75)

a The starting concentration of [35S]sulphate was 0.01 mmol/1. Apparent Ki values (• SE) of different substances in mmol/l. NS, no significant inhibition was observed compared with the controls; > x, an apparent Ki value, if present at all, must be larger than x retool/1. The luminal Ki values for D-lactate were taken from [27]. Further details as stated in the legend to Table 1

rescein, phenol red and bromcresol purple) that we tested against the sulphate transporters behave similarly to the diuretics and probenecid and might have similar effects on the transtubular net transport o f substrates that use the sulphate transporters. There are, however, two dyes, eosine-5-isothiocyanate and b r o m p h e n o l blue, that show n o t only a high inhibitory p o t e n c y against the contraluminal sulphate exchanger but also a g o o d inhibitory p o t e n c y against the luminal N a + - s u l p h a t e cotransporter. It w o u l d be interesting to test the effect o f these substances on sulphate, thiosulphate and oxalate clearance. In conclusion: it was hypothesized that the transport systems at the luminal and contraluminal cell side o f the proximal renal tubule, which are involved in net secretion or net reabsorption, must be p r o f o u n d l y different. H o w ever, it turned o u t that the N a § dependence and the substrate specificity o f the luminal and contraluminal dicarboxylate transport systems are quite similar [21, 29]. A l o n g these lines, it also has been shown that similar Na§ + ion exchangers [3] and p - a m i n o h i p p u r a t e / 2 oxoglutarate exchangers are located at the luminal and the contraluminal cell side [19]. In the present study it was shown that luminal and contraluminal sulphate transporters have the same or similar substrate specificity although the luminal transporter works in c o t r a n s p o r t with N a § and the contraluminal transporter as a H C O 3 exchanger. T h u s the differences between transporters for the same substrate in polar epithelia m a y reside in different driving forces (Na § dependence, H § dependence, electric double layer) or different regulatory events rather than in a p r o f o u n d difference o f molecular structure. A t the present time one might hypothesize that evolution has created closely related families o f transporters n o t only for luminal and contraluminal t r a n s p o r t o f the same substrate, but also for the anion transporters located at the contraluminal cell side with overlapping specificity [24]. Since also the relative inhibitory potencies (app. Ki/Km test substrate) o f different substrates for contraluminal sulphate and p - a m i n o h i p p u r a t e transport are identical, the molecular structure o f these two carriers might also be similar [35]. Cloning o f the different trans-

porters will decide whether this hypothesis is correct or not.

Acknowledgement.We thank Mr. G. Rumrich for expert technicaI help in performing the experiments with simultaneous per•177 of the per• capillaries.

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465 14. L6w I, Friedrich T, Burckhardt G (1984) Properties of an anion exchanger in rat renal basolateral membrane vesicles. Am J Physiol 246: F 334- F 342 15. Lficke H, Stange G, Murer H (1979) Sulphate-ion/sodium-ion co-transport by brush-border membrane vesicles isolated from rat kidney cortex. Biochem J 182 :223 - 229 16. Mudge GH, Berndt WO, Vattin H (1973) Tubular transport of urea, glucose, phosphate, uric acid, sulfate and thiosulfate. In: Handbook of physiology, section 8. Renal physiology. American Physiological Society, Washington, DC, pp 587-652 17. Renfro JL, Pritchard JB (1983) Sulfate transport by flounder renal tubule brush border: presence of anion exchange. Am J Physiol 244 : F488 - F496 18. Samarzija I, Molnar V, Fr6mter E (1981) The stoichiometry of Na § coupled anion absorption across the brushborder membrane of rat renal proximal tubule. In: Takfics L (ed) Advances in physiological sciences, vol 11. Kidney and body fluids. Pergamon, Oxford, pp 419-423 19. Schmitt C, Burckhardt G (1991) Renal p-aminohippurate (PAH) transport systems in rat and bovine kidney exhibit species differences (abstract). Pflfigers Arch 419 : A 120 20. Schneider EG, Durham JC, Sacktor B (1984) Sodium-dependent transport of inorganic sulfate by rabbit renal brush-border membrane vesicles. Biol Chem 259:14591 - 14599 21. Sheridan E, Rumrich G, Ullrich KJ (1983) Reabsorption of dicarboxylic acids from the proximal convolution of rat kidney. Pfltigers Arch 399 : 18 - 28 22. Shimada H, Burckhardt G (1986) Kinetic studies of sulfate transport in basolateral membrane vesicles from rat renal cortex. Pfliigers Arch 407:160-167 23. Turner RJ (1984) Sodium-dependent sulfate transport in renal outer cortical brush border membrane vesicles. Am J Physiol 247 : F 793 - F 798 24. Ullrich KJ, Rumrich G (1988) Contraluminal transport systems in the proximal renal tubule involved in secretion of organic anions. Am J Physiol 254: F 453 - F 462 25. Ullrich KJ (1991) Transport of organic molecules. In: Hatano M (ed) Nephrology, vol II. Springer, Berlin Heidelberg New York, pp 1372-1375

26. Ullrich K J, Rumrich G, K16ss S (1980) Active sulfate reabsorption in the proximal convolution of the rat kidney: specificity, Na + and HCO~- dependence. Pfliigers Arch 383:159-163 27. Ullrich KJ, Rumrich G, K16ss S (1982) Reabsorption ofmonocarboxylic acids in the proximal tubule of the rat kidney: I. Transport kinetics of D-lactate, Na+-dependence, pH-dependence and effect of inhibitors. Pfliigers Arch 395 : 2 1 2 - 219 28. Ullrich K J, Rumrich G, K16ss S, Fasold H (1982) Reabsorption of monocarboxylic acids in the proximal tubule of the rat kidney: III. Specificity for aromatic compounds. Pflfigers Arch 395 : 227- 231 29. Ullrich K J, Fasold H, Rumrich G, K16ss S (1984) Secretion and contraluminal uptake of dicarboxylic acids in the proximal convolution of rat kidney. Pfliigers Arch 400:241 - 2 4 9 30. Ullrich KJ, Rumrich G, K16ss S (1984) Contraluminal sulfate transport in the proximal tubule of the rat kidney: I. Kinetics, effects of K +, Na +, Ca 2+, H +, and anions. Pfl/igers Arch 402: 2 6 4 - 271 31. Ulh'ich KJ, Rumrich G, K16ss S (1985) Contraluminal sulfate transport in the proximal tubule of the rat kidney: II. Specificity: sulfate-ester, sulfonates and amino sulfonates. Pfl~gers Arch 404: 293 - 299 32. Ullrich KJ, Rumrich G, K16ss S (1985) Contraluminal sulfate transport in the proximal tubule of the rat kidney: III. Specificity: disulfonates, di- and tricarboxylates and sulfocarboxylates. Pfliigers Arch 404: 300- 306 33. UUrich K J, Rumrich G, K16ss S (1985) Contraluminal sulfate transport in the proximal tubule of the rat kidney: IV. Specificity: salicylate analogs. Pfliigers Arch 404: 307 - 310 34. Ullrich K J, Rumrich G, K16ss S (1985) Contraluminal sulfate transport in the proximal tubule of the rat kidney: V. Specificity: phenolphthaleins, sulfonphthaleins, and other sulfo dyes, sulfamoyl-compounds and diphenylamine-2-carboxylates. Pflfigers Arch 404: 311 - 318 35. Ullrich KJ, Rumrich G, Fasold H, Zaki L (1987) Affinity labels as substrates for the anion transport systems at the contraluminal cell side of the renal proximal tubule. In: Kovacevic Z, Guder WG (eds) Molecular nephrology. Walter de Gruyter, Berlin, pp 8 5 - 9 0