Reabsorption of betaine in Henle's loops of rat kidney in vivo

Stefan Pummer1, William H. Dantzler2, Yeong-Hau H. Lien3, Gilbert W. Moeckel3, Katharina Völker1, and Stefan Silbernagl1

1 Physiologisches Institut der Universität Würzburg, D-97070 Würzburg, Germany; and Departments of 2 Physiology and 3 Medicine, University of Arizona, Tucson, Arizona 85724


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

This study was designed 1) to localize and 2) to characterize betaine reabsorption from the tubular lumen in rat kidney in vivo, and 3) to test whether reabsorption is modulated by the diuretic state. [14C]betaine (+ [3H]inulin) was microperfused through the proximal convoluted tubule (PCT) and microinfused into late proximal (LP) and early distal (ED) tubules, long loops of Henle (LLH), and vasa recta of the rat in vivo et situ, and the fractional recovery of the 14C label was determined endproximally (PCT) and in the final urine, respectively. [14C]betaine was not reabsorbed during ED microinfusion, whereas fractional reabsorption during LP microinfusion was 82% at 0.06 mM betaine and decreased gradually to 4.8% at 60 mM. L-Proline had lower Michaelis-Menten constant (Km) and sarcosine a higher Km than betaine. Chronic, but not acute, diuresis inhibited betaine reabsorption in Henle's loops. Fractional [14C]betaine reabsorption in PCT was much smaller than that during LP microinfusion. [14C]betaine (7.28 mM) microinfused 1) into LLH was reabsorbed to 30% and 2) into vasa recta appeared in the ipsilateral urine to a much higher extent than contralaterally. In both cases, no saturation was detected at 70 mM. We conclude that betaine is reabsorbed by mediated transport from descending limbs of short Henle's loops by a proline-preferring carrier in a diuresis-modulated manner. In the deep medulla, bidirectional blood/urine betaine transport exists.

betaine transport; rat kidney; organic osmolytes; in vivo microinfusion


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

IN HUMANS 2-N,N,N-trimethylamino acetate or (glycine) betaine is a normal plasma and urine constituent (6, 12). It is well established that betaine functions as an organic osmolyte not only in plants and bacteria but also in the renal medulla, where it helps medullary cells to survive the high osmolality of the medullary environment during antidiuresis (2, 9); even bacteria growing in urine are protected against hypertonicity by taking up urinary betaine (6). In the kidney, betaine is synthesized from choline by choline dehydrogenase and betaine-aldehyde dehydrogenase in renal cortical cells (14, 17, 26). The highest betaine concentrations, however, are found in the renal medulla (3, 30). Water diuresis or furosemide decreased medullary betaine accumulation to a variable extent (3, 20, 30). Madin-Darby canine kidney cells in culture, used as a model for betaine uptake into collecting duct cells (11, 18, 23, 28, 29), take up betaine in a tonicity-dependent manner from the basolateral cell side (28). Therefore, these cells were used as a source for cloning the Na+- and Cl--dependent betain/gamma -aminobutyric acid transporter BGT1 (23, 29). BGT1 mRNA abundance in rats increases in the outer and inner medulla after dehydration (16) and is more than twofold higher in rats treated with an antidiuretic hormone V2-receptor agonist compared with water diuresis (5). Miyai et al. (15) demonstrated that BGT1 mRNA is predominantly present in the thick ascending limb (TAL) of Henle's loop and in inner medullary collecting ducts of rats and that the TAL signal was rapidly increased by a NaCl load, an effect that could be prevented by furosemide. Expression of BGT1 in rat macula densa cells can be reduced by furosemide as well (4).

These data strongly suggest that betaine enters the cells of the inner medullary collecting duct as well as those of the TAL, including the macula densa, by the BGT1 transporter from the basolateral side at a rate that is determined by the local environmental tonicity to some extent. However, the low fractional betaine excretion of <6% (12, 19) shows that filtered betaine is nearly completely reabsorbed; i.e., it can enter renal epithelial cells from the luminal side somewhere along the nephron. Studies with rabbit renal brush-border membrane vesicles showed that betaine can be transported through the luminal membrane of cortical tubule cells by electrogenic Na+- and H+-symporters (27). These transporters might be responsible for reabsorption of filtered betaine.

The main questions we asked in this study were as follows. 1) Where along the nephron and to what extent is betaine reabsorbed in the rat in vivo? 2) What are the kinetics and the specificity of the reabsorption mechanism? 3) Is there any influence of acute or chronic water diuresis on the reabsorption rate of betaine?

To anwer these questions we microinfused 14C-labeled betaine at 1) different concentrations, 2) together with potential competitive transport inhibitors, or 3) during acute or chronic water diuresis, into late proximal (LP) or early distal (ED) tubule sections of superficial nephrons as well as into the long loops of Henle (LLH) of juxtamedullary nephrons and into vasa recta of the rat in vivo et situ and determined the fractional recovery of the 14C label compared with comicroinfused [3H]inulin in the final urine.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Male Munich-Wistar rats weighing between 220 and 320 g [or 70-120 g for LLH and ascending vasa recta (AVR) experiments] were purchased from Charles River, Sulzfeld, Germany, and fed with Altromin Standard Diet 1320. The drinking fluid differed among four groups. Group A (antidiuretic rats) and group B [rats with acutely (after onset of the anesthesia) induced diuresis] had free access to water; group C (4-day chronic diuretic rats) had a 50 g/l sucrose solution as drinking fluid for 4 days; and group D (10-day chronic diuretic rats) had the same sucrose solution for 10 days before the operation day. The diuretic or antidiuretic status was verified during surgery by cryoscopic measurement of urine osmolality (see Table 1).

                              
View this table:
[in this window]
[in a new window]
 
Table 1.   Urine osmolality of experimental groups

The animals were anesthetized with Inactin [Byk-Gulden, Konstanz, Germany; 120 mg/kg body wt (BW)]. A tracheostomy was performed, and polyethylene cannulas were placed in the right jugular vein for infusions. The animals were infused with Ringer solution at a rate of 0.25 ml · min-1 · kg BW-1 (antidiuretic group A and chronic diuretic groups C and D). The acutely diuretic rats of group B were infused with diluted Ringer solution (Ringer/distilled water = 1:2) at 1 ml/min per kg BW. One hundred and twenty minutes after the start of the infusion, mean urine osmolality was 127 mosmol/kgH2O (Table 1). From this time on, microinfusion experiments were performed in group B rats for the next 4 h. The Ringer solution contained the following (in g/l): 9 NaCl, 0.4 KCl, 0.25 CaCl2 · 2H2O, and 0.2 NaHCO3. The kidney was prepared for tubule micropuncture by using standard techniques (1).

Microinfusion into superficial nephrons (LP, ED). After identification of the nephron segments by intravenous injection of Lissamine green SF (Chroma-Gesellschaft, Schmidt, Köngen, Germany) at a dose of 0.02 ml of a 100 g/l solution titrated with NaOH to pH 7.4, the tubule was micropunctured by using glass capillaries. The latter had ground tips (outer tip diameter 9-11 µm) and were mounted on a microperfusion pump (21). Microinfuson sites were (see Fig. 1) 1) the last superficial loop of the proximal tubule (LP) and 2) the first superficial loop of the distal tubule (ED). In all cases, the microinfusate (10 nl/min) added to the endogenous flow rate of tubular fluid. The microinfusate contained (in g/l) 9 NaCl, 0.4 KCl, 0.25 CaCl2 · 2H2O, and 0.5 NaHCO3 as well as 0.2 mg/l [3H]inulin (8.29 GBq/g = 224 mCi/g; DuPont-NEN, Bad Homburg, Germany) and 0.06 mmol/l [14C]betaine (2.04 GBq = 55 mCi/mmol). [14C]betaine was prepared from [14C]choline (Amersham, Arlington Heights, IL) by choline dehydrogenase and purified as described previously (16, 17, 26). Unlabeled betaine, L-alanine, L-proline, sarcosine, or tetramethylammonium was added at the concentrations indicated in RESULTS. Microinfusion lasted for 10 min. Starting shortly before microinfusion, the ipsilateral urine was collected from the ureteral catheter in 15-min fractions for 1 h, and the 14C and 3H disintegrations per minute of each fraction were counted in a liquid scintillation spectrometer (1600 TR, Canberra-Packard, Frankfurt/Main, Germany). As a control, the urine of the contralateral kidney was collected from a bladder catheter in 15-min fractions during the same 1-h period. The [3H]inulin counts (if any) and the [14C]betaine counts in the contralateral urine, never exceeding 10% of those in the ipsilateral urine, were subtracted from the latter. After this correction, the fractional recovery was calculated from the total 14C and 3H disintegrations per minute counts, respectively, of the 1-h collecting period, as shown in Fig. 1.


View larger version (36K):
[in this window]
[in a new window]
 
Fig. 1.   Microinfusion technique. Solutions containing [14C]betaine (14Cinf) and [3H]inulin (3Hinf) were microinfused (10 nl/min for 10 min) by using a microperfusion pump (21) into the last superficial loop of the proximal tubule (late proximal; LP), into the first superficial loop of the distal tubule (early distal; ED), or into long loops of Henle (LLH) near their hairpin bend. Starting shortly before microinfusion, ipsilateral and contralateral urine were collected in 15-min fractions for 1 h, and 14C and 3H disintegrations per minute were counted (14Curine, 3Hurine). For details see MATERIALS AND METHODS.

Microinfusion into LLH. The experiments in LLH (see Fig. 1) were performed as described previously (7). Briefly, the papilla of the left kidney was exposed, and a single ascending limb of a LLH was punctured near the hairpin bend with a glass micropuncture pipette, having an external tip diameter of 5-6 µm, and mounted to a microperfusion pump (21). The tip of this pipette was coated with platinum glaze to make it easily visible (10). The loop was then infused with a TES-buffered (10 mmol/l) solution containing [3H]inulin and [14C]betaine (7.28 mmol/l) as described above. The microinfusion solution also contained Lissamine green (20 g/l) so that the flow in the loop could be seen and so that we could determine whether there was any extravasation from the loop that would make the infusion technically unacceptable. The microinfusion was generally maintained at 10 nl/min. After the microinfusion was well established (usually 2-3 min), collections of urine emerging from the ducts of Bellini were made with a second micropuncture pipette (external tip diameter 12-14 µm). The urinary volume recovery at this collection site was much smaller than that obtained from the ureteral catheter during microinfusion into superficial nephrons. Therefore, the lowest [14C]betaine concentration used had to be higher (7.28 mmol/l) than that used for superficial nephrons (0.06 mmol/l). The radioactivity in the collected fluid and the initial perfusion solution was measured in a liquid scintillation counter (1600 TR, Canberra-Packard) to determine the fractional recovery of the infused [14C]betaine in the pooled urine collecting under oil from all the ducts of Bellini on the ipsilateral side. Two to five collections of approx 70-100 nl were made in each infusion experiment (the number depending on the length of time the infusion could be maintained), and the mean value for the fractional recovery for all collections was used as the value for that microinfusion experiment.

Microinfusions into AVR. The infusions into the AVR were performed in a manner identical to that described above for infusions into ascending loops of Henle. AVR were easily identified by observing the direction of flow of the red blood cells. Because it is relatively easy to puncture and infuse an ascending vas rectum, we could maintain the infusion long enough, as in our previous experiments (8), for the inulin infused to be uniformly distributed and filtered by both kidneys. The usual length of these infusions was 20-25 min, but the inulin appeared to be uniformly distributed (i.e., a steady state appeared to be attained) within the first 5 min (8). As in our previous study (8), we collected urine simultaneously from the ducts of Bellini of the exposed papilla, as described above, and from the contralateral kidney via the bladder cannula.

In determining the amount of betaine relative to inulin appearing in the collections from each kidney (see also Ref. 8), we took into account the fact that at steady state (assuming equal glomerular filtration rates for both kidneys) one-half of the infused inulin should be excreted by each kidney. Thus we took the quotient of betaine-to-inulin ratios in urine vs. infusate ([betaine]/[inulin])urine/([betaine]/[inulin])infusate; brackets indicate concentration) calculated for each kidney and divided this value by two. This gives the fraction of infused betaine (relative to inulin) excreted on each side. The sum of the fractions for the ipsilateral and contralateral kidneys gives the total fraction of the infused betaine (relative to inulin) excreted by both kidneys combined. The difference between the values obtained for the ipsilateral and contralateral kidneys gives the fraction of the microinfused betaine (relative to inulin) secreted on the ipsilateral side. All other aspects of the infusions, as well as collection of urine emerging from the ducts of Bellini and handling of samples, were as described above for infusions into loops of Henle.

Microperfusion of the proximal convoluted tubule. Segments of proximal convoluted tubule were microperfused (21) with a solution containing (in g/l) 5.8 NaCl, 0.37 KCl, 0.22 CaCl2 · 2H2O, 0.42 NaHCO3 and 9.0 D-mannitol at a rate of 20 nl/min. For testing betaine reabsorption, 0.2 mg/l [3H]inulin and 0.06 mmol/l [14C]betaine were added to this solution. Sudan black-stained castor oil was microinjected with a micropipette into the first superficial loop of the proximal convolution, and the endogenous tubule fluid was drained subsequently into the same pipette. The tubule was microperfused with a second micropipette between the second superficial loop (distal end of the oil block) and the last accessible loop of the proximal convolution (perfusion length approx 2-3 mm), where the perfusate was recollected and the fractional late proximal recovery of [14C]betaine was determined.

Chemicals. Betaine, L-alanine, sarcosine and L-proline were purchased from Sigma Chemical, Deisenhofen, Germany;TES from Serva, Heidelberg, Germany; tetramethylammonium from Aldrich, Steinheim, Germany; Lissamine green from Chroma-Gesellschafr, Köngen, Germany; and all other chemicals from Merck, Darmstadt, Germany.

Calculations and statistics. The maximal reabsorption rate Jmax (pmol · s-1 · loop of Henle-1) and the apparent Michaelis-Menten constant (Km; mmol/l) of [14C]betaine reabsorption from short loops of Henle were roughly estimated from the mean reabsorption rates J at the initial concentrations C (see DISCUSSION), whereby
<IT>J</IT> = (1 − recovery) ⋅ microinfusion rate

⋅ C(pmol ⋅ s<SUP>−1</SUP> ⋅ loop of Henle<SUP>−1</SUP>)
by the three linearized plots of the Michaelis-Menten equation 1/J vs. 1/C, C/J vs. C, and J vs. J/C, as well as by nonlinear regression of the J vs. C according to Malo and Berteloot (13).

All results are given as means ± SE (n, number of microinfused or perfused nephron segments). The level of significance for differences between means of unpaired observations was determined with Student's t-test. Differences assessed by t-test were considered statistically significant at P < 0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In a first series of experiments, we microinfused (10 nl/min) a solution containing 0.06 mmol/l [14C]betaine (+ [3H]inulin) into ED tubule sections appearing at the surface of the kidney and determined the fractional excretion of the 14C activity (compared with [3H]inulin) in the final urine of antidiuretic rats (group A; see MATERIALS AND METHODS). It turned out that 99.6 ± 0.28% (n = 7) appeared in the urine. Thus neither the distal convoluted tubule of superficial nephrons nor the collecting duct reabsorbs betaine to any significant extent. For this reason, any reabsorption found during LP microinfusion must have taken place between LP and ED micropuncture sites, i.e., in the short loops of Henle.

In a second series of experiments (group A rats), we microinfused 0.06, 0.6, 6, 30, or 60 mmol/l [14C]betaine (+ [3H]inulin) into LP tubule sections appearing at the surface of the kidney. As can be seen from Table 2, fractional reabsorption (= 1 - fractional excretion) of [14C]betaine decreased from 81.8% at 0.06 mmol/l to 4.8% at 60 mmol/l betaine. Absolute reabsorption rates at the same concentrations increased from 8.2 to 480 fmol · s-1 · loop of Henle-1 (Table 2). From these values, Jmax and Km of [14C]betaine reabsorption in short loops of Henle were roughly estimated by nonlinear regression of the J vs. C values (Jmax = 550 fmol/s; Km = 3.90 mmol/l) as well as by the three linearizations of the Michaelis-Menten equation, i.e., 1/J vs. 1/C (Jmax = 562 fmol/s; Km = 4.05 mmol/l; correlation coefficient r = 0.996), J vs. J/C (Jmax = 541 fmol/s; Km = 3.87 mmol/l; r = 0.996) and C/J vs. C (Jmax = 515 fmol/s; Km = 3.12 mmol/l; r = 0.998). Thus short loops of Henle have a betaine reabsorption capacity of ~515-562 fmol/s. Taking into account that the infusate (10 nl/min) was diluted by the endogenous endproximal tubular flow rate (~15 nl/min; S. Silbernagl, unpublished observations), the Km of [14C]betaine reabsorption in short loops of Henle amounts to ~1.2-1.6 mmol/l. These data demonstrate that betaine reabsorption from short loops of Henle reflects mediated transport.

                              
View this table:
[in this window]
[in a new window]
 
Table 2.   [14C]betaine reabsorption during microperfusion of the proximal convoluted tubule and during late proximal microinfusion

To determine the extent to which betaine is reabsorbed from the proximal convoluted tubule, we microperfused these segments with 0.06 mmol/l [14C]betaine (+ [3H]inulin) by puncturing early to middle proximal tubules and collecting the microperfusate at LP sites. The fractional reabsorption rate turned out to be only 11.5 ± 3.5% (n = 6), reflecting an absolute reabsorption rate of 2.3 ± 0.7 fmol/s in this case.

To evaluate the extent to which [14C]betaine can be reabsorbed from the ascending limb of Henle's loop or enters the tubule or collecting duct lumen from the vasa recta, we microinfused a solution containing 7.28 mmol/l [14C]betaine into the ascending limb of LLH near the hairpin bend or into nearby AVR. The results were as follows. Fractional reabsorption of [14C]betaine (= 1 - fractional recovery; see Fig. 1) amounted to 30 ± 2% (n = 6) during LLH microinfusion, a value not changed (30 ± 2%, n = 3) by a 10-fold increase in betaine concentration (70 mmol/l) in the infusate. These data indicate that the betaine reabsorption during LLH microinfusion, in contrast to the data during LP microinfusion, is not saturable. During AVR microinfusion, recovery of [14C]betaine was 65 ± 5% (n = 7) in the ipsilateral urine and 18 ± 3% (n = 7) in the contralateral urine. Elevating the betaine concentration in the infusate to 70 mmol/l did not significantly change the fractional urinary recovery (61 ± 4%, n = 7 and 13 ± 2%, n = 7, respectively). Thus the ipsilateral blood-to-lumen secretion of [14C]betaine amounts to 47% of the vasa recta load and could not be saturated.

In further sets of experiments (group A rats), we repeated the LP microinfusion of 0.06 mmol/l [14C]betaine in the presence of structurally related compounds, i.e., L-alanine, sarcosine, and tetramethylammonium (6 or 60 mmol/l), as well as L-proline (0.6 or 6 mmol/l). L-Proline inhibited fractional [14C]betaine reabsorption in short loops of Henle (Fig. 2) from the control value of ~82% (Table 2) to 39% (0.6 mmol/l) and to 7.4% (6 mmol/l). As betaine alone is reabsorbed at 0.6 and 6 mmol/l to only 73 and 33%, respectively (Table 2), L-proline has about a sevenfold lower Km at the transporter than does betaine itself. On the other hand, addition of 6 or 60 mmol/l sarcosine resulted in a fractional [14C]betaine reabsorption of 65% (6 mmol/l) or 26% (60 mmol/l). This compares with 33% (6 mmol/l) and 4.8% (60 mmol/l) of betaine alone (Table 2), showing that sarcosine has about a sixfold higher apparent Km at the carrier than betaine. L-Alanine and tetramethylammonium had only weak (but significant) inhibitory effects on [14C]betaine reabsorption in short loops of Henle (Fig. 2).


View larger version (32K):
[in this window]
[in a new window]
 
Fig. 2.   Fractional [14C]betaine reabsorption [= 1 - (fractional 14C recovery in final urine)] ± SE during microinfusion (10 nl/min) of 0.06 mmol/l [14C]betaine into LP segments (n) of superficial nephrons in presence and absence of 0.6, 6, or 60 mmol/l betaine, of 0.6 and 6 mmol/l L-proline, or of 6 and 60 mmol/l sarcosine, L-alanine, or tetramethylammonium (TMA). * P < 0.001.

In further sets of experiments, we investigated whether acute diuresis (group B; see MATERIALS AND METHODS) or chronic diuresis of 4 (group C) or 10 days (group D) influence reabsorption of 0.06 mmol/l [14C]betaine in short loops of Henle. As shown in Fig. 3, the acute diuretic group B rats reabsorbed [14C]betaine to the same extent as the antidiuretic group A rats although the urine osmolality had decreased from 1,600 to 127 mosmol/kgH2O (Table 1) before the microinfusion experiments were started. However, after a water diuresis lasting for 4 or 10 days, fractional [14C]betaine reabsorption in short loops of Henle dropped from 82% (Table 2) to 61 and 52%, respectively, despite a urine osmolality that was higher in these rats (Table 1) than that in group B.


View larger version (13K):
[in this window]
[in a new window]
 
Fig. 3.   Fractional [14C]betaine reabsorption (= 1 - [fractional 14C recovery in final urine]) ± SE during microinfusion (10 nl/min) of 0.06 mmol/l [14C]betaine into LP segments of superficial nephrons in antidiuresis (group A) and acute diuresis (group B) as well as in chronic diuresis lasting for 4 (group C) or 10 days (group D). See MATERIALS AND METHODS for details. * P < 0.001.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Our results show that betaine can be reabsorbed in the convoluted proximal tubule as well as in short loops of Henle of the rat kidney to an extent that matches the whole kidney fractional betaine reabsorption of >94% as determined in humans (12). In our experiments with 0.06 mmol/l betaine, the fractional reabsorption along 2-3 mm of the convolution (11.5%) was much lower than that in the loop of Henle (82%). Extrapolation to the full length of the proximal convoluted tubule (amounting to ~6 mm in rats of this weight; unpublished observations of the authors) indicates that total fractional reabsorption along this segment should not exceed ~30%. Furthermore, the tubular flow rate was certainly not lower in the loop of Henle (10 + about 15 nl/min; see MATERIALS AND METHODS) than in the proximal convoluted tubule (20 nl/min), and (as shown by our LLH results) the saturable betaine reabsorption seems to be restricted to the descending limb of the short loops of Henle, i.e. primarily to the pars recta. As the descending limb is certainly not longer than 6 mm in superficial nephrons (25), our data suggest that the betaine reabsorption capacity per unit length is much higher in the straight portion than in the convoluted portion of the proximal tubule. As this segment is partly located in the outer zone of the renal medulla, betaine reabsorption from the tubule lumen may contribute to the high betaine accumulation (25 mmol/kg wet wt) in the outer medulla of rats observed by Beck et al. (3). Diuresis decreased cellular betaine content to a variable extent (3, 20, 30). Although this is attributable to changes in peritubular uptake of betaine by the BGT1 carrier (see the beginning of this study), our data show that chronic (but not acute) water diuresis also diminishes cellular betaine uptake from the tubule lumen to a significant extent. The reason for this is unknown, but this result could be an explanation for the observation that betaine excretion in humans increases after water loading (19). Luminal uptake of betaine shown in this study is not mediated by the tonicity-regulated BGT1 transporter as this carrier is located in the basolateral membrane (28) and does not accept L-proline (29). The specificity of the carrier reabsorbing betaine shown in this study (affinity for L-proline > betaine > sarcosin >> L-alanine) closely resembles that found for high-affinity proline reabsorption in the rat proximal convolution in vivo (24) as well as that of the intestinal brush-border proline/sodium transporter system found in the brush border of rabbit intestine (22) and renal cortex (27).

Our results also demonstrate that betaine microinfused into ascending loops of Henle is reabsorbed to a significant extent and that this reabsorption could not be saturated with betaine concentrations as high as 70 mmol/l. The route of betaine in this case, as well as that used by betaine from vasa recta to the ipsilateral urine, is unknown. It might reflect nonmediated transport across the thin ascending limb of LLH (3a) as the TAL is a tight epithelium and, according to our results, reabsorption of betaine in the distal convoluted tubule (at least of superficial nephrons) and in the collecting duct does not take place. It might be possible that this transepi- and -endothelium betaine transport in the deep medulla contributes to intercellular betaine distribution in the inner medulla and papilla where organic osmolytes have their toughest job when antidiuresis alternates with diuresis.


    ACKNOWLEDGEMENTS

This study was supported in part by North Atlantic Treaty Organization Collaborative Grant 0104/88, the National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-16294 and DK-45666, and by a grant from the Dialysis Clinic, Inc.


    FOOTNOTES

Parts of this study have been presented as abstracts at the 74th meeting of the Deutsche Physiologische Gesellschaft in Münster, Germany, in 1995; the XIIIth Congress of the International Society of Nephrology in Madrid, Spain, in 1995; and the 127th conference of the Gesellschaft für Biochemie and Molekularbiologie on Transport of Organic Anions and Cations Across Cellular Membranes in Göttingen, Germany, in 1995.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: S. Silbernagl, Physiologisches Institut der Universität Würzburg, Röntgenring 9, D-97070 Würzburg, Germany (E-mail: silbernagl{at}mail.uni-wuerzburg.de).

Received 25 June 1999; accepted in final form 20 October 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Andreucci, V. E. Surgery in the rat. In: Manual of Renal Micropuncture, edited by V. E. Andreucci. Naples, Italy: Idelson, 1978, chapt. 4, p. 50-86.

2.   Beck, F. X., A. Burger-Kentischer, and E. Müller. Cellular response to osmotic stress in the renal medulla. Pflügers Arch. 436: 814-827, 1998[ISI][Medline].

3.   Beck, F. X., M. Schmolke, W. G. Guder, A. Dörge, and K. Thurau. Osmolytes in renal medulla during rapid changes in papillary tonicity. Am. J. Physiol. Renal Fluid Electrolyte Physiol. 262: F849-F856, 1992[Abstract/Free Full Text].

3a.   Brokl, O. H., and W. H. Dantzler. Amino acid fluxes in rat thin limb segments of Henle's loop during in vitro microperfusion. Amer. J. Physiol.Renal Physiol. 277: F204-F210, 1999.

4.   Burger-Kentischer, A., E. Müller, W. Neuhofer, J. März, K. Thurau, and F. X. Beck. Expression of Na+/Cl-/betaine and Na+/myo-inositol transporters, aldose reductase and sorbitol dehydrogenase in macula densa cells of the kidney. Pflügers Arch. 436: 807-809, 1998[ISI][Medline].

5.   Burger-Kentischer, A., E. Müller, W. Neuhofer, J. März, K. Thurau, and F. X. Beck. Expression of aldose reductase, sorbitol dehydrogenase and Na+/myo-inositol and Na+/Cl-/betaine transporter mRNAs in individual cells of the kidney during changes in the diuretic state. Pflügers Arch. 437: 248-254, 1999[ISI][Medline].

6.   Chambers, S. T., and K. M. Kunin. Isolation of glycine betaine and proline betaine from human urine. J. Clin. Invest. 79: 731-737, 1987[ISI][Medline].

7.   Dantzler, W. H., and S. Silbernagl. Amino acid transport: microinfusion and micropuncture of Henle's loops and vasa recta. Am. J. Physiol. Renal Fluid Electrolyte Physiol. 258: F504-F513, 1990[Abstract/Free Full Text].

8.   Dantzler, W. H., and S. Silbernagl. Specificity of amino acid transport in renal papilla: microinfusion of Henle's loops and vasa recta. Am. J. Physiol. Renal Fluid Electrolyte Physiol. 261: F495-F504, 1991[Abstract/Free Full Text].

9.   Garcia-Perez, A., and M. B. Burg. Renal medullary organic osmolytes. Physiol. Rev. 71: 1081-1115, 1991[Abstract/Free Full Text].

10.   Häberle, D. A., J. M. Davis, and G. Mayer. Production of microperfusion pipettes suitable for use with colourless solutions. Pflügers Arch. 376: 191-192, 1978[ISI][Medline].

11.   Handler, J. S., and H. M. Kwon. Regulation of the myo-inositol and betaine cotransporters by tonicity. Kidney Int. 49: 1682-1683, 1996[ISI][Medline].

12.   Lever, M., P. C. B. Sizeland, L. M. Bason, C. M. Hayman, and S. T. Chambers. Glycine betaine and proline betaine in human blood and urine. Biochim. Biophys. Acta 1200: 259-264, 1994[ISI][Medline].

13.   Malo, C., and A. Berteloot. Analysis of kinetic data in transport studies: new insights from kinetic studies of Na+-D-glucose cotransport in human intestinal brush-border membrane vesicles using a fast sampling, rapid filtration apparatus. J. Membr. Biol. 122: 127-141, 1991[ISI][Medline].

14.   Miller, B., H. Schmid, T. J. Chen, M. Schmolke, and W. G. Guder. Determination of choline dehydrogenase activity along the rat nephron. Biol. Chem. Hoppe-Seyler 377: 129-137, 1996[ISI][Medline].

15.   Miyai, A., A. Yamauchi, T. Moriyama, T. Kaneko, M. Takenaka, T. Suriura, H. Kitamura, A. Ando, M. Tohyama, S. Shimada, E. Imai, and T. Kamada. Expression of betaine transporter mRNA: its unique localization and rapid regulation in rat kidney. Kidney Int. 50: 819-827, 1996[ISI][Medline].

16.   Moeckel, G. W., L.-W. Lai, W. G. Guder, H. M. Kwon, and Y.-H. H. Lien. Kinetics and osmoregulation of Na+- and Cl--dependent betaine transporter in rat renal medulla. Am. J. Physiol. Renal Physiol. 272: F100-F106, 1997[Abstract/Free Full Text].

17.   Moeckel, G. W., and Y.-H. H. Lien. Distribution of de novo synthesized betaine in rat kidney: role of renal synthesis on medullary betaine accumulation. Am. J. Physiol. Renal Physiol. 272: F94-F99, 1997[Abstract/Free Full Text].

18.   Nakanishi, T., R. J. Turner, and M. B. Burg. Osmoregulation of betaine transport in mammalian renal medullary cells. Am. J. Physiol. Renal Fluid Electrolyte Physiol. 258: F1061-F1067, 1990[Abstract/Free Full Text].

19.   Nakanishi, T., T. Yamada, T. Iida, O. Uyama, and M. Sugita. Betaine excretion increases by water loading after dehydration. In: Cellular and Molecular Biology of the Kidney, edited by H. Koide, H. Endou, and K. Kurokawa. Basel: Karger, 1991, vol. 95, p. 264-271. (Contrib. Nephrol.)

20.   Schmolke, M., A. Bornemann, and W. G. Guder. Site-specific regulation of organic osmolyts along the rat nephron. Am. J. Physiol. Renal Fluid Electrolyte Physiol. 271: F645-F652, 1996[Abstract/Free Full Text].

21.   Sonnenberg, H., and P. Deetjen. Methode zur Durchströmung einzelner Nephronabschnitte. Pflügers Arch. 268: 669-274, 1964.

22.   Stevens, B. R., and E. M. Wright. Substrate specificity of the intestinal brush-border proline/sodium (IMINO) transporter. J. Membr. Biol. 87: 27-34, 1985[ISI][Medline].

23.   Takenaka, M., S. B. Bagnasco, A. S. Preston, S. Uchida, A. Yamauchi, H. M. Kwon, and J. S. Handler. The canine betaine gamma -amino-n-butyric acid transporter gene: diverse mRNA isoforms are regulated by hypertonicity and are expressed in a tissue-specific manner. Proc. Natl. Acad. Sci. USA 92: 1072-1076, 1995[Abstract].

24.   Völkl, H., and S. Silbernagl. Molecular specificity of tubular reabsorption of L-proline. Pflügers Arch. 387: 253-259, 1980[ISI][Medline].

25.   Wahl, M. Messung der Nettoresorption von Natriumchlorid und Wasser im proximalen Konvolut der Rattenniere (Inaugural dissertation). Munich, Germany: Ludwig-Maximilians-Universität, 1968.

26.   Wirthensohn, G., and W. G. Guder. Studies on renal choline metabolism and phosphatidylcholine synthesis. In: Biochemistry of Kidney Functions, INSERM Symposium No. 21, edited by François Morel. New York: Elsevier Biomedical, 1982, p. 119-128.

27.   Wunz, T. M., and S. H. Wright. Betaine transport in rabbit renal brush-border membrane vesicles. Am. J. Physiol. Renal Fluid Electrolyte Physiol. 264: F948-F955, 1993[Abstract/Free Full Text].

28.   Yamauchi, A., H. M. Kwon, S. Uchida, A. S. Preston, and J. S. Handler. Myo-inositol and betaine transporters regulated by tonicity are basolateral in MDCK cells. Am. J. Physiol. Renal Fluid Electrolyte Physiol. 261: F197-F202, 1991[Abstract/Free Full Text].

29.   Yamauchi, A., S. Uchida, H. M. Kwon, A. S. Preston, R. B. Robey, A. Garcia-Perez, M. B. Burg, and J. S. Handler. Cloning of a Na+- and Cl--dependent betaine transporter that is regulated by hypertonicity. J. Biol. Chem. 267: 649-652, 1992[Abstract/Free Full Text].

30.   Yancey, P. H., and M. B. Burg. Distribution of major organic osmolytes in rabbit kidneys in diuresis and antidiuresis. Am. J. Physiol. Renal Fluid Electrolyte Physiol. 257: F602-F607, 1989[Abstract/Free Full Text].


Am J Physiol Renal Physiol 278(3):F434-F439
0363-6127/00 $5.00 Copyright © 2000 the American Physiological Society