Active urea transport in the rat inner medullary collecting duct: Functional characterization and initial expression cloning

Kidney International, Vol. 49 (1996), pp. 1611—1614

Active urea transport in the rat inner medullary collecting duct: Functional characterization and initial expression cloning JEFF M. SANDS, S0NIA MARTIAL, and TAIsuI IsozAKE Renal Division, Department of Medicine, Emory University School of Medicine, Atlanta, Georgia, USA

Active urea transport in the rat inner medullary collecting duct:

inability to measure the urea gradient across the collecting duct in

Functional characterization and initial expression cloning. Active transport of urea has been proposed to exist in the inner medullary collecting duct (IMCD) of low-protein fed mammals for over 30 years. We perfused IMCD subsegments from rats fed a standard (18%) or a low (8%) protein diet and tested for the presence of active urea transport. We found no

vivo, and thus could not definitively prove whether urea was

active urea transport in terminal IMCDs, regardless of diet. In initial

[23], may be useful." Since the proposed experiment was not performed over for 25 years, we decided to perfuse initial and terminal IMCDs microdissected from rats maintained on low

IMCDs from rats fed 18% protein or fed 8% protein for one to two weeks, we again found no active urea transport. However, in rats fed 8% protein for three to four weeks, we found significant net urea reabsorption. This active urea reabsorption was inhibited when Na,KtATPase activity was inhibited by adding 1 mM ouabain or removing bath potassium, suggesting a secondaiy active transport process. Removing sodium from the perfus-

ate completely inhibited net urea reabsorption, demonstrating that this active urea transport is dependent upon the presence of sodium in the tubule lumen. Unlike the facilitated urea transporter, the active urea transporter was not inhibited by phloretin nor stimulated by vasopressin,

actively reabsorbed. As Ullrich et a! wrote in 1967, to prove active urea transport in the IMCD, "the in vivo microperfusion method

for the isolated collecting duct, recently published by Burg et al

(8%) protein diets and on standard (18%) protein diets to determine whether active urea transport was indeed induced by a low-protein diet. After determining that active urea transport did exist, we performed studies to investigate the nature of this active urea transport process and performed initial studies to clone its eDNA.

suggesting that it is a distinct transport protein. To test this hypothesis, we

size-separated poly(A)-RNA prepared from inner medullae of rats fed 8% protein for three weeks and injected it into Xenopus laevis oocytes.

Demonstration of active urea transport To determine whether active urea transport could be demonRNA from a 4.4 to 8.4 kb size fraction increased urea permeability fourfold compared to water-injected oocytes or injecting RNA from other strated in isolated perfused IMCDs, rats were fed either an 18% size-fractions. We conclude that feeding rats a low-protein diet for three or 8% protein diet (NIH-31 or NIH-31 M, respectively, Ziegler weeks induces the expression of an unique, secondary active, sodiumBrothers, Gardner, PA, USA). Rats fed this low-protein diet grow dependent urea transporter whose eDNA is between 4.4 and 8.4 kb in size. and maintain normal values of serum albumin, total protein, and In addition, our results suggest that it will be possible to clone the eDNA creatinine [24, 25]. Initial or terminal IMCDs were dissected and for this sodium-urea cotransporter by expression in Xenopus laevis oocytes. perfused at 37°C using standard techniques [24, 26, 27]. To measure net urea transport, tubules were perfused with identical perfusate and bath solutions containing 3 m urea [24, 25, 28]. In all mammals studied, including human [1], camel [2], sheep In initial IMCDs from rats fed 18% protein, there was no net [3—7], and rat [8—19], low-protein diets change inner medullary urea flux (1 1 pmol/mm/min, N = 4, P = NS [241). Similarly, urea content. The fractional excretion of urea is significantly there was no net urea flux in initial IMCDs from rats fed 8% reduced in rats fed a low-protein diet, consistent with an increase protein for one or two weeks (1 week: 1 0.4 pmol/mm/min, N = in tubular reabsorption of urea [11, 12, 14, 15, 20]. In addition, the 4; 2 weeks: 1 1 pmol/mm/min, N = 5 [251). However, there was normal inner medullary urea concentration profile is reversed in a significant net transepithelial urea flux in initial IMCDs from low-protein fed rats [15—17]. The maximum inner medullary urea rats fed 8% protein for three or four weeks (3 weeks: 5 1 concentration is found in the base of the inner medulla and pmol/mm/min, N 5, P < 0.02 [25]; 4 weeks: 5 1 pmol/mm/min, decreases towards the papillary tip [15—171. These data raise the N = 13, P < 0.002 [24]; Fig. 1). possibility that a low-protein diet might induce active urea transIn terminal IMCDs from rats fed 18% protein, there was no net port [15—17, 211 in the base of the inner medulla, presumably in urea flux (0 0.3 pmol/mm/min, N = 4, P = NS, [24]). Unlike the initial inner medullary collecting duct (IMCD) which is initial IMCDs, there was also no net urea flux in terminal IMCDs located in this region of the kidney. from rats fed 8% protein for four weeks (1 0.2 pmol/mm/min, Papillary micropuncture studies performed in rats fed a low- N = 4, P NS, [24]). Thus, feeding rats a low-protein diet induces protein diet are consistent with the hypothesis that urea may be active urea transport in the initial IMCD but not in the terminal actively reabsorbed from the collecting duct [8—10, 13, 18, 20, 22]. IMCD. Unfortunately, these micropuncture studies were limited by the Mechanism of active urea transport To determine whether this active urea transport was primary or secondary active transport, initial IMCDs from rats fed 8% © 1996 by the International Society of Nephrology 1611

1612

Sands et al: Active urea transport in rat initial IMCDs

7

Table 1. Comparison of rat kidney urea transporters

P<0.02

P<0.02

Facilitated urea transporter

Property

Natdependent Vasopressin Phloretin

0

E4 0

Thiourea Expression in IMCD

>(

No [30] Stimulates [24, 271 Inhibits [24, 29] Inhibits [29, 31]

segments Terminal IMCD Yes [27] Initial IMCD 18% Protein diet No [24, 27] 8% Protein diet Yes, after 2 weeks [25] Size of mRNA 2.9 and 4.0 kb [30, 31]

zl ci)

0

Secondary active urea transporter Yes [28] No effect [28] No effect [24, 28]

Not tested No [24] No [24] Yes, after 3 weeks [251 4.4—8.4 kb (current study)

18% Protein 1 Week 2 Weeks 3 Weeks 4 Weeks 8% Protein diet Table 2. Characteristics of active urea transport processes Fig. 1. Net urea flux (pmol/mm/min) in initial !MCDs from rats fed 18% protein or 8% protein for 1, 2, 3, orfbur weeks. Data are mean SE; N is indicated at the bottom of each column. Data are redrawn from [24, 25].

protein

for three weeks were perfused and Na,K-ATPase

activity was inhibited by adding 1 mtvi ouabain to the bath or by removing bath potassium (and replacing it with sodium). Net urea

reabsorption was reversibly inhibited by 57 5% (N = 5, P < 0.01) by adding ouabain (1 m, Sigma) to the bath and by 68

6% (N = 6, p < 0.01 [28]) by removing bath potassium. It was also reversibly inhibited by cooling the tubule to 23°C [24]. Thus, net urea transport was a secondary active transport process. Next, we tested whether net urea transport was dependent upon sodium by replacing perfusate sodium with N-methyl-D-glucamine. When sodium was removed from the perfusate, net urea transport was completely and reversibly inhibited (with Na5, 13 I pmol/mm/min; without Nat, 0 I pmol/mm/min, N = 5, P < 0.01, [28]). However, net urea flux was unchanged when sodium was removed from the bath [28]. Thus, feeding rats a low-protein

Animal

Tissue

Squalus acanthias

Renal tubule

Bufo bufo Rana esculenta Bufo viridis

Skin Skin Skin

Bufo marinus Rabbit (NZW) Rat (SD/LPD)

Skin

PST

Characteristics

References

Sodium-linked (Phioretin-sensitive

not tested) Sodium-independent Sodium-independent Sodium-independent Phloretin-sensitive Sodium-independent Phoretin-sensitive

Initial IMCD Phloretin-insensitive Sodium-dependent

[32]

[33, 34] [33, 351 [33] [361 [371

[38] [24, 28] [281

Abbreviations are: NZW, New Zealand white; PST, proximal straight

tubule; SD/LPD, Sprague-Dawley rats fed 8% protein for 3 weeks; IMCD, inner medullary collecting duct. This Table modified from [28].

phloretin-addition and with sodium-removal suggest the presence of a unique urea transport process in initial IMCDs from rats fed a low-protein diet.

diet induced expression of a previously unknown, secondary

Expression of sodium-dependent active urea transport in oocytes whether it was stimulated by vasopressin or inhibited by phioretin. To investigate whether the secondary active, sodium-dependent Net urea transport was unchanged by adding 10 nM AVP to the urea transporter is an unique transport protein, we have begun bath [28] or by adding 0.25 m'vi phioretin to the perfusate [24, 28]. studies to clone this sodium-urea cotransporter using the Xenopus These properties further distinguish the sodium-urea cotrans- laevis expression cloning method. Oocytes were hand-dissected porter from the facilitated urea transporter that is stimulated by from ovarian fragments of Xenopus laevis, treated with 2 mg/mI collagenase to remove follicular cells, and incubated in Barth's vasopressin [27] and inhibited by phioretiri (Table 1) [24, 29]. solution (88 mivi NaCI, 1 ms KCI, 0.8 mist MgSO4, 0.4 mM Comparison to other active urea transport processes Ca(N03)2, 7.5 mcvi Tris, 2.4 mcvi NaHCO3; pH 7.6) containing 100 Schmidt-Nielsen, Truniger and Rabinowitz [32] first proposed U/ml penicillin and 0.01 mg/mI streptomycin at 18°C (39]. The sodium-linked urea transport in the renal tubule of the spiny next day, the healthy oocytes were injected with 50 nI of water or dogfish Squalus acanthias (Table 2). These studies were based on poly(A)5 RNA (I mg/ml) and incubated in Barth's solution with clearance measurements and they were unable to test the effect of antibiotics at 18°C for three to five days [39]. phloretin [32]. In our studies, the combination of sodium-depenSince sodium-urea cotransport has only been shown to be dence and phloretin-insensitivity in the initial IMCD of rats fed a expressed in the initial IMCD of rats fed 8% protein for at least low-protein diet differentiate this active urea transporter from all three weeks [25, 28], poly(A) RNA was prepared from inner medullae of these rats. Total RNA was isolated from the renal previously described active urea transport processes [33—38]. Bufo active, sodium-dependent urea transporter.

To characterize this sodium-urea cotransporter, we tested

bufo, Rana esculenta, Bufo viridis, and Bufo marinus have sodium-

independent and/or phloretin-sensitive active urea transport pro-

inner medullae of several rats using Tn-Reagent (Molecular Research Center, Inc., Cincinnati, OH, USA). PoIy(A) RNA

cesses in their skins (Table 2). Kawamura and Kokko [38] was purified by oligo(dT)-cellulose chromatography [40].

reported a phioretin-sensitive, active urea secretory process in Oocytes were injected with 50 ng of poly(A)4 RNA or water rabbit proximal straight tubules (Table 2). Thus, our results with and 14C-urea fluxes were measured in the presence of 1 mcvi

1613

Sands et a!: Active urea transport in rat initial IMCDs

phioretin. Phioretin was added because it inhibits 14C-urea flux mediated by UT2 [30] and transepithelial urea transport mediated by the facilitated urea transporter [24, 28, 29], but has no effect on sodium-urea cotransport [28]. 3H-raffinose was added as a control for oocyte integrity; oocytes with > fivefold increase in raffinose permeability were discarded [39]. Oocytes were pre-incubated for

(P
C.)

ci)

E 0 '1

four hours in Barth's solution plus 1 m urea at 18°C, then incubated in Barth's solution plus 150 mrvt NaCl, 1 m urea, 2 iCi of 14C-urea, 5 j.Ci of 3H-raffinose, and 1 mivi phloretin at 18 to

:54

20°C under slow shaking. After incubation, urea uptake was

E

stopped with 3 ml of ice-cold Barth's solution plus 150 mivi NaCl

ci)

and 1 m urea and washed twice with the same solution. Each

cci

oocyte was dissolved in 100 pi of formic acid and the 14C-urea was assayed by liquid scintillation counting [39].

cci

ci)

a-

I1i

3

3

7

Water Fl F2 F3 F4 F5 Poly(A) RNA injected oocytes had a significantly higher urea Poly(A RNA size fractions 0.2 X 10—6 cm/see) than water injected permeability (1.2 oocytes (0.6 0.1 X 10' cm/see). Next, urea permeability was Fig. 2. Urea permeability (10_6 cm/see) in Xenopus laevis oocytes injected measured in the absence of sodium. When oocytes were incubated with 50 nl of water or 5 ng of poly(A) + RNA from the inner medulla of rats in the absence of sodium, there was no significant difference in fed 8% protein for three weeks. Poly(A) RNA size-fractions are: Fl, < kb; F2, 0.24 to 1.8 kb; F3, 1.8 to 4.4 kb; F4, 4.4 to 8.4 kb, and F5, > urea permeability between poly(A) -injected and water-injected 0.24 8.4 kb. Data are mean SE; N is indicated at the bottom of each column. oocytes, consistent with a sodium-dependent urea transporter. Next, the poly(A) RNA was size-separated using agarose gel electrophoresis. PoIy(A) RNA (20 to 40 g) was denatured by References

heating (2 mm at 70° C) and rapidly cooled on ice, then size-separated using a mid-size 0.75% low-melting point agarose

gel. After separation, the fractions were recovered under UV light, the agarose was melted at 70° C, and poly(A) + RNA was recovered from the aqueous phase after centrifugation. The poly(A) RNA fractions were precipitated with ethanol and solubilized in water at a concentration of 0.1 .tg/ml for subsequent injection into oocytes [39, 41]. Oocytes were injected with 50 nl of water or 5 ng of poly(A) RNA of the following sizes: <0.24 kb, 0.24 to 1.8 kb, 1.8 to 4.4 kb, 4.4 to 8.4 kb, and > 8.4 kb. Oocytes injected with poly(A) RNA

in the size-fraction 4.4 to 8.4 kb had a fourfold higher urea permeability (P < 0.02) than water injected oocytes or oocytes injected with poly(A) RNA from the other size-fractions (Fig. 2). Thus, oocytes injected with poly(A) + mRNA from the inner medulla of rats fed 8% protein for three weeks express sodiumdependent urea transport, which suggests that it will be possible to clone a sodium-urea cotransporter eDNA using the oocyte expres-

sion cloning technique. Additionally, the poly(A) RNA size fraction which gave the highest urea permeability, 4.4 to 8.4 kb, is different from the size of rat and rabbit UT2 (2.9 to 4.0 kb) [30,

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