1 Physiologisches Institut der Universität Würzburg, D-97070 Würzburg, Germany; and 2 Department of Physiology, College of Medicine, University of Arizona, Tucson, Arizona 85724-5051
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ABSTRACT |
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Filtered myo-inositol, an important renal intracellular organic osmolyte, is almost completely reabsorbed. To examine tubule sites and specificity and, thus possible mechanism of this reabsorption, we microinfused myo-[3H]inositol or D-[3H]glucose into early proximal (EP), late proximal (LP), or early distal tubule sections of superficial nephrons and into long loops of Henle (LLH) of juxtamedullary nephrons and papillary vasa recta in rats in vivo et situ and determined urinary fractional recovery of the 3H label compared with comicroinfused [14C]inulin. To determine the extent to which the proximal convoluted tubule (PCT) alone contributes to myo-inositol reabsorption, we also microperfused this tubule segment between EP and LP puncture sites. We examined specificity of reabsorptive carrier(s) by adding high concentrations of other polyols and monosaccharides to the infusate. The results show that >60% of the physiological glomerular load of myo-inositol can be reabsorbed in the PCT and >90% in the short loop of Henle (SLH) by a saturable, phloridzin-sensitive process. myo-Inositol can also be reabsorbed in the ascending limb of LLH and can move from papillary vasa recta blood into ipsilateral tubular structures. Essentially no reabsorption occurred in nephron segments beyond the SLH or in collecting ducts. Specificity studies indicate that reabsorption probably occurs via a luminal Na+-myo-inositol cotransporter.
myo-inositol transport; D-glucose transport; sodium-myo-inositol cotransporter
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INTRODUCTION |
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MYO-INOSITOL
(MR = 180) has at least
two important functions in mammalian cells. First, it is a
phosphoinositide precursor and, therefore, plays an integral role in
phospholipase C-mediated and other signal transduction
pathways. Second, it is an intracellular organic osmolyte in the kidney
(especially in the outer medulla) and several other tisues (12,
13, 17, 18, 20, 26, 37, 38). In rats, myo-inositol
plasma concentration amounts to ~50 µmol/l, and
myo-inositol concentrations of >20, 15, and <5 mmol/l have
been found in cell water in rat thick ascending limbs, collecting
ducts, and proximal tubules, respectively (23). Similar
values have been reported for the rabbit kidney (37), where the concentration of myo-inositol in the outer medulla
decreased from ~35 (in antidiuresis) to ~25 mmol/kg wet wt (in
water diuresis). The high myo-inositol concentration
gradient between cytosol and extracellular fluid is maintained by a
Na+-myo-inositol cotransporter (SMIT)
(15). In collecting duct-derived Madin-Darby canine kidney
(MDCK) cells (21a), a hypertonic environment induced an increased
transcription of the SMIT gene and, at the same time, a dramatically
increased cytosolic myo-inositol concentration (34,
36). Of this uptake, 90% occurred at the basolateral cell side
(34). In rat kidney, SMIT was found to be strongly expressed in the medulla and, at lower levels, in the cortex
(35). In situ hybridization revealed that SMIT is
predominantly present in the medullary and cortical thick ascending
limbs of Henle's loop and macula densa cells (35).
Fractional excretion of myo-inositol by the rat kidney amounts to 1-2% (6). Thus highly effective transporter(s) must exist in the luminal membrane of the renal tubules. Takenawa et al. (28) investigated myo-inositol transport in a cortical plasma membrane preparation of rat kidney. They found an uptake mechanism specific for myo-inositol and scyllitol that did not accept D-glucose or D-galactose. In a similar study, Hammerman et al. (11) examined myo-inositol uptake into rabbit renal cortical brush-border membrane vesicles. They found that myo-inositol uptake was electrogenic and saturable as well as stimulated by a electrochemical Na+ gradient. Uptake was inhibited by phloridzin and, to a moderate extent, by D-glucose.
In the present study, we investigated the localization and kinetics of tubular myo-inositol reabsorption in rat kidney and characterized the specificity of the transport mechanism to identify the carrier type(s) involved in tubular myo-inositol reabsorption. For this purpose, we microinfused myo-inositol or D-[3H]glucose into early proximal (EP), late proximal (LP), or early distal (ED) tubule sections of superficial nephrons as well as into long loops of Henle (LLH) of juxtamedullary nephrons of the rat in vivo et situ and determined the fractional recovery of the 3H label compared with comicroinfused [14C]inulin in the final urine. To determine the extent to which the proximal convoluted tubule (PCT) alone contributes to myo-inositol reabsorption, we also microperfused this tubule segment between EP and LP puncture sites. For any reabsorption of myo-[3H]inositol or D-[3H]glucose found, we then examined the specificity of the carrier(s) involved by adding high concentrations of other polyols and monosaccharides to the infusate or perfusate. To elucidate whether myo-inositol in medullary plasma has access to the ipsilateral lumen of collecting ducts, we also microinfused myo-[3H]inositol or D-[3H]glucose together with [14C]inulin into ascending vasa recta and determined the fractional recovery of the 3H label (compared with comicroinfused [14C]inulin) in the ipsilateral and contralateral urine.
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MATERIALS AND METHODS |
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Male Munich-Wistar rats were used in the following three
experimental groups: group A [EP, LP, and ED experiments;
see Figs. 2-4, 6, and 7; mean body weight 178-326 g (mean wt:
270 g), purchased from Simonsen Laboratories, Gilroy, CA];
group B [PCT microinfusion experiments; see Figs. 8 and 9;
body weight 192-475 g (mean wt: 366 g), purchased from
Medizinische Hochschule, Hannover, Germany]; and group C
[LLH experiments; body weight 61-146 g (mean wt: 99 g),
purchased from Medizinische Hochschule]. Group A was fed on Teklad 4% Mouse/Rat Diet 7001; groups B and C
were fed on an Altromin Standard Diet 1320. All groups had free access
to water. The animals were anesthetized with Inactin (Byk-Gulden,
Konstanz, Germany; 120 mg/kg body wt). 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
0.05 ml · min1 · kg
body wt
1 for the larger animals (groups A and
B) and 0.02 ml · min
1 · kg
body wt
1 for the smaller animals (group C).
The Ringer contained the following (in g/l): 9 NaCl, 0.4 KCl, 0.25 CaCl2, and 0.2 NaHCO3. The kidney was prepared
for tubule micropuncture using standard techniques (1).
Microinfusion into superficial nephrons (EP, 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 using glass capillaries (see
Fig. 1). The latter had ground tips
(outer tip diameter 9-11 µm) and were mounted on a
microperfusion pump (25). Microinfuson sites were (see
Fig. 1) 1) the first superficial loop of the proximal tubule
(EP), 2) the last superficial loop of the proximal tubule
(LP), and 3) the first superficial loop of the distal tubule
(ED). In all these cases, the microinfusate (10 nl/min) added to the
endogenous flow rate of tubular fluid. The microinfusate (pH 6.7)
contained (in mmol/l) 154 NaCl, 5.4 KCl, 1.7 CaCl2, 9.6 MOPS; 88 MBq/g (= 2.4 mCi/g) [14C]inulin (NEN,
Perkin- Elmer Life Science, Boston, MA); 10 µmol/l 3H-labeled myo-inositol (2.04 GBq = 55 mCi/mmol, American Radiolabeled Chemicals, St. Louis, MO), or 10 µmol/l 3H-labeled D-glucose (2.23 GBq/mmol = 60 mCi/mmol, American Radiolabeled Chemicals), as well
as unlabeled sugars, polyols, or phloridzin, as indicated in
RESULTS (see Figs. 6 and 7). Microinfusion lasted for 10 min. Starting shortly before microinfusion, the ipsilateral urine was
collected from the ureteral catheter in 30-min fractions for 1 h,
and the 14C and 3H disintegrations per minute
(dpm) counts of each fraction were determined in a liquid scintillation
spectrometer (Beckman LS 6000SE, Anaheim, CA, or Canberra-Packard 1600 TR, Frankfurt/Main, Germany). As a control, the urine of the
contralateral kidney was collected from a bladder catheter in 30-min
fractions during the same 1-h period. The [14C]inulin
counts (if any) and the 3H 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 (see Fig. 1) was calculated from the sum of the
14C and 3H dpm counts, respectively, of the 1-h
collecting period (14Curine and
3Hurine; see Fig. 1) and from the
14C and 3H dpm counts of the microinfusion
solution (14Cinf and
3Hinf; see Fig. 1). For the latter purpose, the
10-min output of the microinfusion pump was collected in a drop of
water.
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Microinfusion into LLH.
The experiments on LLH were performed as described previously (4,
5). 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 (25). The tip of this pipette was coated with platinum glaze to make it
easily visible (9). The loop was then infused with a
solution containing [14C]inulin and
myo-[3H]inositol (90 µM) or
D-[3H]glucose (33 µmol/l) as described
above as well as (in mmol/l) 154 NaCl, 5.4 KCl, 1.7 CaCl2,
2.4 NaHCO3, and 10 TES. The microinfusion solution also
contained lissamine green (20 g/l) so that the flow in the loop could
be seen and 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) (see Fig.
1). 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
myo-[3H]inositol concentration used had to be
higher (90 µmol/l) than that used for superficial nephrons (10 µmol/l). The same consideration applied to the concentration of
D-[3H]glucose (33 vs. 10 µM). 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
myo-[3H]inositol in the urine of the ducts of
Bellini. Two to five collections of 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.
Microinfusion into ascending vasa recta. The infusions into the ascending vasa recta (AVR) were performed in a manner identical to that described above for infusions into ascending loops of Henle. AVR were easily indentified by observing the direction of flow of the red blood cells. As in our previous study (4), during the constant infusion of a vas rectum, 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. 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 (4), for the inulin infused to be uniformly distributed and filtered by both kidneys. Within the first 5 min, inulin appeared to be uniformly distributed in the animal (i.e., a steady state appeared to be attained) so that the amounts obtained from the ipsilateral and contralateral kidneys were equal (4). They remained equal as long as the constant infusion was maintained. Also, once this point was attained, the amounts of the other substance infused (e.g., myo-inositol) collected from each kidney remained constant over time as long as the constant infusion was maintained (4). The usual length of these infusions was 20-25 min.
In determining the amount of myo-inositol or D-glucose relative to inulin appearing in the collections from each kidney (see also Ref. 4), we took into account the fact that at steady state (assuming equal glomerular filtration rates of both kidneys) one-half of the infused inulin should be excreted by each kidney. Thus we divided by two the quotient of the myo-inositol-to-inulin ratios in the urine vs. infusate ([myo-inositol]/[inulin])urine/([myo-inositol]/[inulin])infusate. This gives the fraction of microinfused myo-inositol (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 myo-inositol (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 myo-inositol (relative to inulin) secreted on the ipsilateral side. The same approach was used for D-glucose microinfusions into the AVR. All other aspects of the infusions, as well as the collection of urine emerging from the ducts of Bellini and the handling of samples, were as described above for infusions into the LLH.Microperfusion of the PCT.
Segments of PCT were microperfused (25) at a rate of 20 nl/min with the following solution (in g/l) 6.2 NaCl, 0.37 KCl, 0.22 CaCl2, and 2.2 TES and titrated to pH 7.4. For this
purpose, 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 2-3 mm), where the perfusate was recollected
and the fractional late proximal recovery of
myo-[3H]inositol or
D-[3H]glucose was determined.
Chemicals. myo-Inositol, D-glucose, D-galactose, D-fructose, D-mannose, 3-O-methylglucose, L-fucose, and phloridzin were purchased from Sigma (Deisenhofen, Germany); TES and MOPS from Serva (Heidelberg, Germany); and all other chemicals from Merck (Darmstadt, Germany).
Calculations and statistics.
The maximal reabsorption rate Jmax (pmol/s) and
the apparent Michaelis constant (mmol/l) of
myo-[3H]inositol reabsorption from short loops
of Henle were roughly estimated from the individual reabsorption rates
J at the respective concentrations at the LP microinfusion
site (CLP), where the microinfusion rate of 10 nl/min was
added to the endogenous flow rate of tubular fluid. The latter was also
assumed to amount to 10 nl/min because the single-nephron filtration
rate in rats is ~30 nl/min and late proximal TF/P inulin in rats is
~3. Thus the microinfusate was diluted ~1:1; i.e., CLP
was in fact ~50% of the concentration in the microinfusion solutions
(CMI). The individual values of J and
CLP were used to obtain a least square fit (Sigmaplot 4, Jandel Scientific) of the Michaelis-Menten equation J = Jmax · CLP/(KM + CLP), whereby J = (1 fractional recovery) · microinfusion rate · CMI
[pmol · s
1 · short
loop of Henle
1].
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RESULTS |
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Because we wanted to find out where along the nephron and to what
extent myo-inositol is reabsorbed, we microinfused a
solution containing myo-[3H]inositol and
[14C]inulin into superficial tubular puncture sites and
determined the fractional recovery (see MATERIALS AND
METHODS) of the 3H-label of
myo-inositol compared with the comicroinfused
[14C]inulin. As the reabsorptive process rather than
excretion is the major focus of this paper, most of the following
results are discussed as fractional reabsorption (%) [=100 fractional recovery (%)] (Figs. 2, 3, and 6-9).
Localization, saturation, phloridzin sensitivity, and pH dependence of myo-inositol reabsorption along the nephron. In a first series of experiments, we microinfused (10 nl/min) a solution containing 10 µmol/l 3H-labeled myo-inositol into EP, LP and ED tubule sections appearing at the surface of the kidney. As can be seen from Fig. 2, fractional reabsorption of the 3H label of myo-inositol was 95 ± 1.4% during EP, 96 ± 0.8% during LP, but not significantly different from zero (3.7 ± 5.2%, n = 7) during ED microinfusion. Thus regardless of whether myo-[3H]inositol was injected at EP or LP sites, reabsorption was virtually complete. In contrast, no reabsorption at all was observed between the ED microinfusion site of these superficial (short) nephrons and the final ipsilateral urine. Phloridzin (0.1 mmol/l) added to the microinfusate nearly completely blocked reabsorption of 10 µmol/l myo-[3H]inositol during EP and LP microinfusion. All these microinfusion solutions were buffered to a pH of 6.7. To test whether a higher pH influences the rate of reabsorption, we repeated the LP experiment with 10 µmol/l myo-[3H]inositol at pH 7.6. However, the fractional reabsorption (96 ± 0.31%, n = 8) did not change at all.
Next, we increased the myo-inositol concentration in the microinfusate to 1, 3, 10 and 50 mmol/l (pH 6.7) by adding the nonlabeled compound. As shown in Fig. 2, fractional reabsorption decreased more as the concentration was increased; i.e., transport became saturated. Whereas at 10 µmol/l and 1 mmol/l, fractional reabsorption was found to have nearly the same high value at EP and LP microinfusion sites, at 3 mmol/l, fractional reabsorption during LP microinfusion was only 60% of that during EP microinfusion. At 50 mmol/l, fractional reabsorption amounted to ~15%; i.e., it became nearly fully saturated. As expected, fractional reabsorption during ED microinfusion was still not significantly different from zero at myo-inositol concentrations of 1 (7.12 ± 2.45%, n = 4) and 10 mmol/l (4.99 ± 2.45, n = 4). At 50 mmol/l myo-inositol, the significance level was just reached (PLocalization, saturation, and phloridzin dependence of D-glucose reabsorption along the nephron. To obtain insight into the molecular specificity of myo-inositol vs. D-glucose reabsorption in the loop of Henle (see below), we first had to characterize D-glucose reabsorption in experiments similar to those used with myo-inositol. First, we determined the fractional reabsorption of 10 µmol/l 3H-labeled D-glucose during EP, LP, and ED microinfusion and obtained the following values: 88.7 ± 2.2 (EP, n = 5), 91.3 ± 2.6 (LP, n = 6), and 5.1 ± 0.4% (ED, n = 6). Thus the high fractional reabsorption of D-glucose determined during LP microinfusion reflects nearly exclusively reabsorption in short loops of Henle, because reabsorption beyond the ED site is very small. At higher initial D-glucose concentrations in the LP microinfusate (3, 10, and 50 mmol/l), fractional D-glucose reabsorption amounted to 95.7 ± 2.9 (n = 6), 74.8 ± 4.9 (n = 3), and 35.5 ± 4.9% (n = 5; see Fig. 7), respectively. Thus the reabsorption process saturated. In the presence of 1 mmol/l phloridzin, the fractional reabsorption of 10 µmol/l D-[3H]glucose decreased to 9.5 ± 1.6 (EP, n = 6) and to 12.5 ± 2.4% (LP, n = 7; see Fig. 7). Therefore, D-glucose reabsorption in short loops of Henle was not only saturable but also sensitive to phloridzin.
When we microinfused a solution containing 33 µmol/l 3H-labeled D-glucose plus [14C]inulin into the ascending limb of an LLH near the hairpin bend, 31 ± 8% (n = 10) of D-[3H]glucose was reabsorbed between the puncture site and the urine emerging from the ipsilateral ducts of Bellini. This fraction did not change significantly when 0.1 mmol/l phloridzin or 50 mmol/l nonlabeled D-glucose was added to the microinfusate (see Fig. 3). Thus D-glucose is able to leave the lumen of tubule segments that are situated downstream from the LLH microinfusion site. As ED microinfusion of D-[3H]glucose resulted only in a very small fractional reabsorption, the collecting duct does not seem to be involved in the reabsorptive process. Thus the D-[3H]glucose microinfused into LLH must have been reabsorbed in the ascending limb of Henle's loop of juxtamedullary nephrons. Microinfusing the same solution (33 µmol/l D-[3H]glucose plus [14C]inulin) into vasa recta running parallel to the LLH, we found that fractional recovery was 25 ± 4% (n = 4) in the ipsilateral urine and 5 ± 1% (n = 4) in the contralateral urine. These values did not change significantly when 0.1 mmol/l phloridzin or 50 mmol/l nonlabeled D-glucose was added to the microinfusate. Thus D-[3H]glucose is also able to enter the tubular urine from the ipsilateral vasa recta blood.Molecular specificity of myo-inositol vs. D-glucose
reabsorption in the loop of Henle.
In further sets of experiments, we evaluated the molecular specificity
of the carrier(s) involved in tubular
myo-[3H]inositol reabsorption during LP
microinfusion. For this purpose, 50 mmol/l of the following compounds
were added to the microinfusate containing 10 µmol/l
3H-labeled myo-inositol plus
[14C]inulin: nonlabeled myo-inositol,
scyllo-inositol, D-chiro-inositol, L-chiro-inositol (see Fig.
5), D-fructose,
D-mannose, L-fucose (=6-deoxy-L-galactose), 3-O-methyl-glucose
(=3-O-methyl-D-glucopyranose), D-glucose, D-galactose, and
-methyl-D-glucoside. As can be seen from Fig.
6, scyllo-inositol and
D-chiro-inositol had nearly the same large
inhibitory effect as nonlabeled myo-inositol itself, whereas
L-chiro-inositol, D-glucose,
D-galactose, and
-methyl-D-glucoside had a
much smaller but significant inhibitory effect on
myo-[3H]inositol reabsorption. No inhibition
occurred in the presence of the remaining four compounds.
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Reabsorption of myo-inositol in the PCT.
As can be seen from Fig. 2, fractional reabsorption of
myo-inositol at an initial concentration of 1 and 3 mmol/l
was higher during EP than during LP microinfusion. This means that the
tubule segment located between these two microinfusion sites, i.e., the PCT, contributes to renal myo-inositol reabsorption. To
study this process in more detail directly, we microperfused the
segment between EP and LP micropuncture sites with a solution
containing 10 µmol/l 3H-labeled myo-inositol
(+ [14C]inulin) in the absence and presence of 0.1 mmol/l
phloridzin or 50 mmol/l nonlabeled myo-inositol,
D-glucose, L-fucose, and 3-O-methylglucose at a microperfusion rate of 20 nl/min. The
fractional recovery of the 3H activity was determined in
the perfusate collected at LP micropuncture sites. As shown in Fig.
8, fractional reabsorption of 10 µmol/l myo-inositol was 63.3 ± 3.7% (control) and decreased
to 9.2 ± 2.3, 36.3 ± 2.2, or 17.3 ± 2.4% in the
presence of 50 mmol/l of nonlabeled myo-inositol,
D-glucose, or 0.1 mmol/l phloridzin, respectively.
L-Fucose and 3-O-methylglucose did not have a
significant effect. Qualitatively, these results resemble those for the
short loops of Henle (see Fig. 6), but fractional reabsorption was
generally lower in the microperfused segment of the proximal
convolution than that in short loops of Henle. The microperfusion
solution had a higher pH than that of the LP microinfusion experiments (see MATERIALS AND METHODS). However, this
cannot be the reason for the quantitative difference in reabsorption,
because myo-inositol reabsorption was pH independent in this
pH range.
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Reabsorption of D-glucose in the PCT.
In additional EP microinfusion experiments, we found that fractional
reabsorption of D-glucose at an initial concentration of 50 mmol/l (70.1 ± 4.0%, n = 5) was much higher than
during the LP microinfusion reported above (35.5 ± 4.8%,
n = 5). This last finding was not unexpected and
indicates that the PCT contributes a major part to renal
D-glucose reabsorption. To test the influence of
myo-inositol and other compounds on proximal
D-glucose reabsorption directly, we microperfused the
segment between EP and LP micropuncture sites with a solution
containing 10 µmol/l 3H-labeled D-glucose (+ 14C-inulin) in the absence and presence of 0.1 and 1.0 mmol/l phloridzin or 50 mmol/l nonlabeled D-glucose,
myo-inositol, L-fucose, or 3-O-methylglucose at a microperfusion rate of 20 nl/min.
As shown in Fig. 9, fractional
reabsorption of 10 µmol/l D-glucose was 74.1 ± 2.8% (control) and decreased to 15.7 ± 2.5% in the presence of
50 mmol/l nonlabeled D-glucose and to 16.5 ± 1.4 and
8.0 ± 3.3.% in the presence of 0.1 and 1 mmol/l phloridzin,
respectively. L-Fucose and 3-O-methylglucose had
a small effect, whereas 50 mmol/l myo-inositol did not
influence D-glucose reabsorption at all. Qualitatively,
these results again mirror those from the short loops of Henle (see
Fig. 7), but fractional reabsorption is generally lower in the
microperfused segment of the proximal convolution than in the short
loops of Henle.
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DISCUSSION |
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It has been known for about 50 years that myo-inositol is nearly completely reabsorbed in the mammalian kidney (6). However, only very little has been known about the localization of the reabsorptive process along the nephron and the transport mechanism and its molecular specifity. We investigated these topics by microinfusing and microperfusing single tubule segments of rat kidney in vivo and in situ. Our results indicate that >60% of the physiological glomerular load of myo-inositol can be reabsorbed in the PCT. Moreover, the short loop of Henle alone was able to reabsorb >90% of a myo-inositol load that is higher than the physiological glomerular load. In both segments, myo-inositol reabsorption was phloridzin sensitive and saturable. These findings are in accord with earlier in vitro results showing that renal cortical brush-border membrane vesicles (BBMV) from rat and rabbit take up myo-inositol by a saturable, phloridzin-sensitive process that is stimulated by an electrochemical Na+ gradient (11, 27).
Essentially, no myo-inositol reabsorption occurred in nephron segments beyond the short loops of Henle and in the collecting ducts. This means that reabsorptive data obtained during LP microinfusions represent reabsorption in short loops of Henle. Our data obtained by microinfusing the ascending limb of LLH revealed that about one-half of the microinfused myo-[3H]inositol was reabsorbed downstream from this micropuncture site. The reabsorption must take place in the ascending limb of LLH because the collecting ducts are not able to reabsorb myo-inositol (see above). Moreover, myo-[3H]inositol microinfused into inner medullary AVR appeared in the ipsilateral final urine to a much greater extent than in the contralateral urine. This transport out of the vasa recta was saturable and sensitive to phloridzin. These results taken together suggest that myo-inositol is able to enter medullary cells from the blood side as well as from the tubular lumen. We cannot exclude the possibility that myo-inositol could be secreted in the connecting tubule in the medullary ray, but we do not think that it is likely.
In these studies, we have shown that myo-inositol reabsorption in the short loops of Henle and in the PCT is nearly completely saturated if a high concentration of nonlabeled myo-inositol is added to the microinfusate. To test the specificity of this transport and, at the same time, to get an idea of which apical carrier(s) is involved in tubular myo-inositol reabsorption, we tried to inhibit it with high concentrations of several polyols and sugars. We observed a strong inhibition of myo-inositol reabsorption when it was infused together with the myo-inositol derivatives scyllo-inositol (or scyllitol) or D-chiro-inositol. Scyllo-inositol has previously been shown to inhibit strongly myo-inositol uptake into rat BBMV (27). Moreover, scyllo-inositol induces nearly the same steady-state current as myo-inositol in oocytes into which cRNA of canine SMIT has been injected (10).
As illustrated in Fig. 6, D-fructose, D-mannose, L-fucose, and 3-O-methylglucose did not have any effect on myo-inositol reabsorption. Thus it is very unlikely that myo-inositol is reabsorbed by the fructose uniporter GLUT5 or a mannose symporter (7, 24).
We tested L-fucose and 3-O-methylglucose because previous studies have shown that uptake currents in canine SMIT-injected oocytes are high with L-fucose but very low with 3-O-methylglucose (10). In the same paper, uptake current was also measured in oocytes transfected with rabbit Na+-glucose luminal transporter 1 (SGLT1). In these cells, 3-O-methylglucose was very well accepted by SGLT1 but L-fucose not at all (10). Our results, i.e., that neither of the two substances influenced myo-inositol transport in the short loop of Henle (Fig. 6) or the PCT (Fig. 8), could mean that myo-inositol is not reabsorbed by either SMIT or SGLT1. However, although SMIT from the dog and SMIT from the rat have a 95% homology at the protein level (358 amino acids in both), dog cDNA (2,870 bases) is much longer than rat cDNA (1,155 bases) (15, 17, 35). Therefore, the specificity of canine SMIT might be quite different from that of the rat because splice variants of SMIT have been found even in the same species (8, 22). Thus we cannot rule out the possibility that SMIT is responsible for the reabsorption of myo-inositol in the rat kidney.
However, are the D-glucose transporters in tubule apical
membranes involved in myo-inositol reabsorption? Tubular
D-glucose reabsorption shows axial heterogeneity
(29). SGLT1 is present in the S3 segment and SGLT2, in the
S1 and S2 segments of the proximal tubule (14, 16).
Substrates of the rat and rabbit SGLT1 are D-glucose,
-methyl-D-glucoside, D-galactose, and
3-O-methyl-D-glucose (10, 16);
substrates of the rabbit and pig SGLT2 are D-glucose and
-methyl-D-glucoside, but not D-galactose and
3-O-methyl-D-glucose (14, 19).
However, localization and/or specificity of these transporters does not
seem to be clear cut because D-galactose is reabsorbed in
the PCT (32), which does not include the S3 segment. In
the present study, D-glucose,
-methyl-D-glucoside, and D-galactose, but
not 3-O-methyl-D-glucose, had a small inhibitory effect on myo-inositol reabsorption in short loops of Henle
(Fig. 6). The effect of D-glucose was higher in the PCT
(Fig. 8), where the low-affinity SGLT2 transporter is located.
Small-to-moderate inhibition of myo-inositol uptake by
D-glucose also has been shown in rat and rabbit BBMV
(11, 27). Thus it is possible that myo-inositol
is reabsorbed by the SGLT carriers. However, D-glucose uptake currents in oocytes transfected with canine SMIT are small and
not different for D- and L-glucose. This
observation hardly seems compatible with the hypothesis that SGLTs
represent the apical myo-inositol transporters in the kidney
because glucose transport by SGLTs is highly stereospecific.
To further test the hypothesis that myo-inositol is reabsorbed via one of the SGLT carriers, we characterized D-glucose reabsorption in the short loops of Henle (Fig. 7) and the PCT (Fig. 9) in the same way as we did myo-inositol reabsorption. In both segments, marked D-glucose reabsorption, which was phloridzin sensitive and saturable, took place. The roughly estimated kinetic constants were similar to those determined in earlier microperfusion experiments in vivo (2). However, a comparison with the kinetic constants for myo-inositol reabsorption also obtained in the present study shows that Jmax and Km for D-glucose reabsorption in the short loops of Henle are about fourfold higher than those for myo-inositol reabsorption.
D-Glucose reabsorption was inhibited to a moderate extent
by -methyl-D-glucoside in the short loop of Henle and by
3-O-methyl-D-glucose in the PCT. However, in the
context of this paper, it is most important that neither
myo-inositol nor D-chiro-inositol
influenced D-glucose reabsorption in either segment to any
extent. These results clearly show that the SGLT carriers do not accept
myo-inositol to a significant extent, thereby confirming
earlier results that showed that myo-inositol does not have
any influence on radiolabeled
-methyl-D-glucoside uptake
in SGLT1- (16) or SGLT2-transfected oocytes
(14).
A further possibility is that tubular reabsorption of myo-inositol is mediated by the H+-myo-inositol symporter (HMIT) expressed predominantly in the mammalian brain (31) but apparently to a small extent also in the kidney. Increasing the driving force for H+ uptake into oocytes by decreasing the extracellular pH from 7 to 5 increased myo-inositol transport roughly sixfold (31). However, in the present work, increasing the pH of the microinfusate from 6.7. to 7.6 did not have any effect on myo-inositol reabsorption in the short loops of Henle. Thus it seems to be unlikely that the tubular reabsorption investigated in this paper is mediated by HMIT. The fact that the uptake of myo-inositol into tubular BBMV is driven by a Na+ gradient (10, 25) also speaks against this possibility and also against tubular reabsorption of myo-inositol by a uniporter like GLUT5.
Most recently, an orphan cDNA 43% identical in sequence to SMIT [now called SMIT1 (3)] was expressed in oocytes that were subsequently voltage clamped (3). Inward currents were found during superfusion with myo-inositol, D-chiro-inositol, and, to a smaller extent, with D-glucose. Uptake by this transporter (called SMIT2 by the authors) exhibited stereospecificity for D-glucose and D-chiro-inositol. L-Fucose was not accepted by SMIT2. This specificity resembles not only that found earlier when myo-inositol uptake was studied in liver cells (21) but also that of our present data. Thus we hypothesize that SMIT2 is responsible for renal tubular reabsorption of myo-inositol.
We conclude from our data that tubular reabsorption of myo-inositol in the PCT and in the loop of Henle is responsible for the nearly complete fractional reabsorption of this compound. Myo-inositol reabsorption is not mediated by the SGLTs, the HMIT carrier, the mannose transporter, or the GLUT5 uniporter. Our data support our hypothesis that myo-inositol reabsorption across the luminal membrane of the PCT, the short loop of Henle, and the ascending limb of the LLH is mediated by SMIT2. The extent to which this luminal route is used for the high myo-inositol accumulation in the cells of the thick ascending limb of Henle's loop (23) and for renal intracellular inositol metabolism remains to be elucidated.
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ACKNOWLEDGEMENTS |
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We thank Olga Brokl and Kristen Evans for encouragement and support in the laboratory at the University of Arizona and Dr. S. H.Wright for valuable discussions.
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FOOTNOTES |
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This study was supported in part by the Alexander von Humboldt Foundation and National Institute of Diabetes and Digestive and Kidney Diseases Research Grant DK-16294.
Parts of this study have been presented as abstracts at the PharmaConference 2001 in Interlaken, Switzerland, at the 34th Congress of the International Union of Physiological Sciences in Christchurch, New Zealand (Abstract CD-ROM, 2001), and at the 81st Congress of the Deutsche Physiologische Gesellschaft in Tübingen, March 2002 (Pflügers Arch Suppl 433: S215, 2002).
Address for reprint requests and other correspondence: S. Silbernagl, Physiologisches Institut der Universität Würzburg, Röntgenring 9, D-97070 Würzburg, Germany (E-mail: stefan.silbernagl{at}mail.uni-wuerzburg.de).
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. Section 1734 solely to indicate this fact.
10.1152/ajprenal.00395.2002
Received 6 November 2002; accepted in final form 13 February 2003.
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