Tubular reabsorption of myo-inositol vs. that ofD-glucose in rat kidney in vivo - 《American Journal of Physiology》美国生理学杂志干细胞之家



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发表于 2009-4-21 13:38 |只看该作者 |倒序浏览 |打印

作者：StefanSilbernagl, KatharinaVölker,  William H.Dantzler作者单位：1 Physiologisches Institut der UniversitätWürzburg, D-97070 Würzburg, Germany; and Department of Physiology, College of Medicine,University of Arizona, Tucson, Arizona 85724-5051 ' Y1 P' W" r$ H4 v
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      【摘要】7 V9 B) s2 z) v! `
   Filtered myo -inositol, animportant renal intracellular organic osmolyte, is almost completelyreabsorbed. To examine tubule sites and specificity and, thus possiblemechanism of this reabsorption, we microinfused myo -[ 3 H]inositol or D- [ 3 H]glucose into early proximal (EP), lateproximal (LP), or early distal tubule sections of superficial nephronsand into long loops of Henle (LLH) of juxtamedullary nephrons andpapillary vasa recta in rats in vivo et situ and determined urinaryfractional recovery of the 3 H label compared withcomicroinfused [ 14 C]inulin. To determine the extent towhich the proximal convoluted tubule (PCT) alone contributes to myo -inositol reabsorption, we also microperfused this tubulesegment between EP and LP puncture sites. We examined specificity ofreabsorptive carrier(s) by adding high concentrations of other polyolsand monosaccharides to the 60% ofthe physiological glomerular load of myo 90% in the short loop of Henle (SLH) by asaturable, phloridzin-sensitive process. myo -Inositol canalso be reabsorbed in the ascending limb of LLH and can move frompapillary vasa recta blood into ipsilateral tubular structures.Essentially no reabsorption occurred in nephron segments beyond the SLHor in collecting ducts. Specificity studies indicate that reabsorptionprobably occurs via a luminal Na - myo -inositol cotransporter.
      【关键词】 myo inositol transport D glucose transport sodium myo inositol cotransporter
   INTRODUCTION" B! W; I! G( c3 V1 D
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MYO -INOSITOL ( M R = 180) has at leasttwo important functions in mammalian cells. First, it is aphosphoinositide precursor and, therefore, plays an integral role inphospholipase C -mediated and other signal transductionpathways. 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 -inositolplasma concentration amounts to ~50 µmol/l, and myo 20, 15, and limbs, collectingducts, and proximal tubules, respectively ( 23 ). Similarvalues have been reported for the rabbit kidney ( 37 ), where the concentration of myo -inositol in the outer medulladecreased from ~35 (in antidiuresis) to ~25 mmol/kg wet wt (inwater diuresis). The high myo -inositol concentrationgradient between cytosol and extracellular fluid is maintained by aNa - myo -inositol cotransporter (SMIT)( 15 ). In collecting duct-derived Madin-Darby canine kidney(MDCK) cells (21a), a hypertonic environment induced an increasedtranscription of the SMIT gene and, at the same time, a dramaticallyincreased 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 ispredominantly present in the medullary and cortical thick ascendinglimbs of Henle's loop and macula densa cells ( 35 ).
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Fractional excretion of myo -inositol by the rat kidneyamounts to 1-2% ( 6 ). Thus highly effectivetransporter(s) must exist in the luminal membrane of the renal tubules.Takenawa et al. ( 28 ) investigated myo -inositoltransport in a cortical plasma membrane preparation of rat kidney. Theyfound an uptake mechanism specific for myo -inositol andscyllitol that did not accept D -glucose or D -galactose. In a similar study, Hammerman et al.( 11 ) examined myo -inositol uptake into rabbitrenal cortical brush-border membrane vesicles. They found that myo -inositol uptake was electrogenic and saturable as wellas stimulated by a electrochemical Na gradient. Uptake wasinhibited by phloridzin and, to a moderate extent, by D -glucose.

In the present study, we investigated the localization and kinetics oftubular myo -inositol reabsorption in rat kidney and characterized the specificity of the transport mechanism to identify the carrier type(s) involved in tubular myo -inositolreabsorption. For this purpose, we microinfused myo -inositolor D- [ 3 H]glucose into early proximal (EP),late proximal (LP), or early distal (ED) tubule sections of superficialnephrons as well as into long loops of Henle (LLH) of juxtamedullarynephrons of the rat in vivo et situ and determined the fractionalrecovery of the 3 H label compared with comicroinfused[ 14 C]inulin in the final urine. To determine the extentto which the proximal convoluted tubule (PCT) alone contributes to myo -inositol reabsorption, we also microperfused this tubulesegment between EP and LP puncture sites. For any reabsorption of myo -[ 3 H]inositol or D- [ 3 H]glucose found, we then examined thespecificity of the carrier(s) involved by adding high concentrations ofother polyols and monosaccharides to the infusate or perfusate. Toelucidate whether myo -inositol in medullary plasma hasaccess to the ipsilateral lumen of collecting ducts, we alsomicroinfused myo -[ 3 H]inositol or D -[ 3 H]glucose together with[ 14 C]inulin into ascending vasa recta and determinedthe fractional recovery of the 3 H label (compared withcomicroinfused [ 14 C]inulin) in the ipsilateral andcontralateral urine.

MATERIALS AND METHODS

Male Munich-Wistar rats were used in the following threeexperimental 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 fromMedizinische 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 accessto 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 forinfusions. The animals were infused with Ringer solution at a rate of0.05 ml · min 1 · kgbody wt 1 for the larger animals ( groups A and B ) and 0.02 ml · min 1 · kgbody wt 1 for the smaller animals ( group C ).The Ringer contained the following (in g/l): 9 NaCl, 0.4 KCl, 0.25 CaCl 2, and 0.2 NaHCO 3. The kidney was preparedfor tubule micropuncture using standard techniques ( 1 )./ }  D! _. n& X

Microinfusion into superficial nephrons (EP, LP, ED). After identification of the nephron segments by intravenous injectionof lissamine green SF (Chroma-Gesellschaft, Schmidt, Köngen,Germany) at a dose of 0.02 ml of a 100-g/l solution titrated with NaOHto pH 7.4, the tubule was micropunctured using glass capillaries (seeFig. 1 ). The latter had ground tips(outer tip diameter 9-11 µm) and were mounted on amicroperfusion pump ( 25 ). Microinfuson sites were (seeFig. 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 theendogenous flow rate of tubular fluid. The microinfusate (pH 6.7)contained (in mmol/l) 154 NaCl, 5.4 KCl, 1.7 CaCl 2, 9.6 MOPS; 88 MBq/g (= 2.4 mCi/g) [ 14 C]inulin (NEN,Perkin- Elmer Life Science, Boston, MA); 10 µmol/l 3 H-labeled myo -inositol (2.04 GBq = 55 mCi/mmol, American Radiolabeled Chemicals, St. Louis, MO), or 10 µmol/l 3 H-labeled D -glucose (2.23 GBq/mmol = 60 mCi/mmol, American Radiolabeled Chemicals), as wellas 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 wascollected from the ureteral catheter in 30-min fractions for 1 h,and the 14 C and 3 H disintegrations per minute(dpm) counts of each fraction were determined in a liquid scintillationspectrometer (Beckman LS 6000SE, Anaheim, CA, or Canberra-Packard 1600 TR, Frankfurt/Main, Germany). As a control, the urine of thecontralateral kidney was collected from a bladder catheter in 30-minfractions during the same 1-h period. The [ 14 C]inulincounts (if any) and the 3 H counts in the contralateralurine, never exceeding 10% of those in the ipsilateral urine, weresubtracted from the latter. After this correction, the fractionalrecovery (see Fig. 1 ) was calculated from the sum of the 14 C and 3 H dpm counts, respectively, of the 1-hcollecting period ( 14 C urine and 3 H urine; see Fig. 1 ) and from the 14 C and 3 H dpm counts of the microinfusionsolution ( 14 C inf and 3 H inf; see Fig. 1 ). For the latter purpose, the10-min output of the microinfusion pump was collected in a drop ofwater.

Fig. 1. Microinfusion technique. Solutions containing myo -[ 3 H]inositol or D -[ 3 H]glucose ( 3 H inf )and [ 14 C]inulin ( 14 C inf ) weremicroinfused (10 nl/min for 10 min) by a microperfusion pump( 23 ) into the first superficial loop of the proximaltubule [early proximal (EP)], into the last superficial loop of theproximal tubule [late proximal (LP)], into the first superficial loopof the distal tubule [early distal (ED)], and into long loops ofHenle (LLH) near their hairpin bend. Starting shortly beforemicroinfusion, the ipsilateral urine was collected from the ipsilateralureter in 30-min fractions for 1 h, and the 3 H( 3 H urine ) and 14 C( 14 C urine ) disintegrations/min (dpm) werecounted. For details, see MATERIALS AND METHODS.- R4 K' d8 q9 c# [' ?5 i; A
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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 asingle ascending limb of a LLH was punctured near the hairpin bend witha glass micropuncture pipette, having an external tip diameter of5-6 µm, and mounted to a microperfusion pump ( 25 ). The tip of this pipette was coated with platinum glaze to make iteasily visible ( 9 ). The loop was then infused with asolution containing [ 14 C]inulin and myo -[ 3 H]inositol (90 µM) or D -[ 3 H]glucose (33 µmol/l) as describedabove as well as (in mmol/l) 154 NaCl, 5.4 KCl, 1.7 CaCl 2,2.4 NaHCO 3, and 10 TES. The microinfusion solution alsocontained lissamine green (20 g/l) so that the flow in the loop couldbe seen and we could determine whether there was any extravasation fromthe loop that would make the infusion technically unacceptable. Themicroinfusion was generally maintained at 10 nl/min. After themicroinfusion was well established (usually 2-3 min), collectionsof urine emerging from the ducts of Bellini were made with a secondmicropuncture pipette (external tip diameter 12-14 µm) (see Fig. 1 ). The urinary volume recovery at this collection site was muchsmaller than that obtained from the ureteral catheter duringmicroinfusion into superficial nephrons. Therefore, the lowest myo -[ 3 H]inositol concentration used had to behigher (90 µmol/l) than that used for superficial nephrons (10 µmol/l). The same consideration applied to the concentration of D -[ 3 H]glucose (33 vs. 10 µM). Theradioactivity in the collected fluid and the initial perfusion solutionwas measured in a liquid scintillation counter (1600 TR,Canberra-Packard) to determine the fractional recovery of the infused myo -[ 3 H]inositol in the urine of the ducts ofBellini. Two to five collections of 70-100 nl were made in eachinfusion experiment (the number depending on the length of time theinfusion could be maintained), and the mean value for the fractionalrecovery for all collections was used as the value for thatmicroinfusion experiment.! r' w. l$ j7 U, C" X

Microinfusion into ascending vasa recta. The infusions into the ascending vasa recta (AVR) were performed in amanner identical to that described above for infusions into ascendingloops of Henle. AVR were easily indentified by observing the directionof flow of the red blood cells. As in our previous study( 4 ), during the constant infusion of a vas rectum, wecollected urine simultaneously from the ducts of Bellini of the exposedpapilla, as described above, and from the contralateral kidney via thebladder cannula. Because it is relatively easy to puncture and infusean ascending vas rectum, we could maintain the infusion long enough, asin our previous experiments ( 4 ), for the inulin infused tobe uniformly distributed and filtered by both kidneys. Within the first5 min, inulin appeared to be uniformly distributed in the animal (i.e.,a steady state appeared to be attained) so that the amounts obtainedfrom the ipsilateral and contralateral kidneys were equal( 4 ). They remained equal as long as the constant infusionwas maintained. Also, once this point was attained, the amounts of theother substance infused (e.g., myo -inositol) collected fromeach kidney remained constant over time as long as the constantinfusion was maintained ( 4 ). The usual length of theseinfusions was 20-25 min.7 w0 A* L! ]9 r; {  H5 x' J! v- ^

In determining the amount of myo -inositol or D -glucose relative to inulin appearing in the collectionsfrom each kidney (see also Ref. 4 ), we took into accountthe fact that at steady state (assuming equal glomerular filtrationrates of both kidneys) one-half of the infused inulin should beexcreted by each kidney. Thus we divided by two the quotient of the myo -inositol-to-inulin ratios in the urine vs. infusate([ myo -inositol]/) urine /([ myo -inositol]/) infusate. Thisgives the fraction of microinfused myo -inositol (relative toinulin) excreted on each side. The sum of the fractions for theipsilateral and contralateral kidneys gives the total fraction of theinfused myo -inositol (relative to inulin) excreted by both kidneys combined. The difference between the values obtained for theipsilateral and contralateral kidneys gives the fraction of themicroinfused 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 ofthe infusions, as well as the collection of urine emerging from theducts of Bellini and the handling of samples, were as described abovefor infusions into the LLH.6 i; F# y9 i7 I! V
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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 CaCl 2, and 2.2 TES and titrated to pH 7.4. For thispurpose, Sudan black-stained castor oil was microinjected with amicropipette into the first superficial loop of the proximalconvolution, and the endogenous tubule fluid was drained subsequentlyinto the same pipette. The tubule was microperfused with a secondmicropipette between the second superficial loop (distal end of the oilblock) and the last accessible loop of the proximal convolution(perfusion length 2-3 mm), where the perfusate was recollectedand the fractional late proximal recovery of myo -[ 3 H]inositol or D -[ 3 H]glucose was determined.
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Chemicals. myo -Inositol, D -glucose, D -galactose, D -fructose, D -mannose,3- O -methylglucose, L -fucose, and phloridzin werepurchased 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 J max (pmol/s) andthe apparent Michaelis constant (mmol/l) of myo -[ 3 H]inositol reabsorption from short loopsof Henle were roughly estimated from the individual reabsorption rates J at the respective concentrations at the LP microinfusionsite (C LP ), where the microinfusion rate of 10 nl/min wasadded to the endogenous flow rate of tubular fluid. The latter was alsoassumed to amount to 10 nl/min because the single-nephron filtrationrate 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., C LP was in fact ~50% of the concentration in the microinfusion solutions(C MI ). The individual values of J andC LP were used to obtain a least square fit (Sigmaplot 4, Jandel Scientific) of the Michaelis-Menten equation J = J max · C LP /( K M C LP ), whereby J = (1 fractional recovery) · microinfusion rate · C MI [pmol · s 1 · shortloop of Henle 1 ].

Because there was virtually no myo -[ 3 H]inositol reabsorption downstream ofthe ED microinfusion site (see RESULTS ), fractional reabsorption during LP microinfusion is equivalent to that in shortloops of Henle.! ^* t2 [, ]6 y( j
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All results shown in Figs. 2-4 and 6-9 are means ± SE( n = no. of microinfused or microperfused nephronsegments). The level of significance fordifferences between means of unpaired observations was determined withStudent's t -test. Differences assessed by t- testwere considered statistically significant at P
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Fig. 2. Fractional 3 H reabsorption [=100 3 H fractional recovery in the final urine (in %)] duringmicroinfusion (10 nl/min) of 0.01, 1, 3, 10, or 50 mmol/l myo -[ 3 H]inositol into EP, LP, and ED segmentsof superficial nephrons. Values are means ± SE; the no. ofmicroinfused nephron segments is in parentheses.
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Fig. 3. Fractional 3 H reabsorption [=100 fractional recovery of 3 H in the ipsilateral final urine(in %)] during microinfusion (10 nl/min) of 90 µmol/l myo -[ 3 H]inositol into long loops of Henle inthe presence or absence of 0.1 mmol/l phloridzin or 50 mmol/lnonlabeled myo -inositol. Values are means ± SE; theno. of microinfused nephron segments is in parentheses.: K  e6 y- m! h6 A. t; @0 R

Fig. 4. Fractional 3 H recovery in the ipsilateral andcontralateral final urine during microinfusion (10 nl/min) of 90 µmol/l myo -[ 3 H]inositol into ascending vasarecta in the presence or absence of 0.1 mmol/l phloridzin or 50 mmol/lnonlabeled myo -inositol. Values are means ± SE; theno. of microinfused nephron segments is in parentheses." V* W3 W, f0 E% j$ ^- J. |! x
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RESULTS( N4 ~, m- p# J

Because we wanted to find out where along the nephron and to whatextent myo -inositol is reabsorbed, we microinfused asolution containing myo -[ 3 H]inositol and[ 14 C]inulin into superficial tubular puncture sites anddetermined the fractional recovery (see MATERIALS AND METHODS ) of the 3 H-label of myo -inositol compared with the comicroinfused[ 14 C]inulin. As the reabsorptive process rather thanexcretion is the major focus of this paper, most of the followingresults are discussed as fractional reabsorption (%) [=100 fractional recovery (%)] (Figs. 2, 3, and 6-9 ).
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Localization, saturation, phloridzin sensitivity, and pH dependenceof myo-inositol reabsorption along the nephron. In a first series of experiments, we microinfused (10 nl/min) asolution containing 10 µmol/l 3 H-labeled myo -inositol into EP, LP and ED tubule sections appearing atthe surface of the kidney. As can be seen from Fig. 2, fractional reabsorption of the 3 H label of myo -inositol was95 ± 1.4% during EP, 96 ± 0.8% during LP, but notsignificantly different from zero (3.7 ± 5.2%, n = 7) during ED microinfusion. Thus regardless of whether myo -[ 3 H]inositol was injected at EP or LPsites, reabsorption was virtually complete. In contrast, noreabsorption at all was observed between the ED microinfusion site ofthese superficial (short) nephrons and the final ipsilateral urine.Phloridzin (0.1 mmol/l) added to the microinfusate nearly completelyblocked reabsorption of 10 µmol/l myo -[ 3 H]inositol during EP and LPmicroinfusion. All these microinfusion solutions were buffered to a pHof 6.7. To test whether a higher pH influences the rate ofreabsorption, we repeated the LP experiment with 10 µmol/l myo -[ 3 H]inositol at pH 7.6. However, thefractional reabsorption (96 ± 0.31%, n = 8) didnot change at all.
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Next, we increased the myo -inositol concentration in themicroinfusate to 1, 3, 10 and 50 mmol/l (pH 6.7) by adding thenonlabeled compound. As shown in Fig. 2, fractional reabsorptiondecreased more as the concentration was increased; i.e., transportbecame saturated. Whereas at 10 µmol/l and 1 mmol/l, fractionalreabsorption was found to have nearly the same high value at EP and LPmicroinfusion sites, at 3 mmol/l, fractional reabsorption during LPmicroinfusion was only 60% of that during EP microinfusion. At 50 mmol/l, fractional reabsorption amounted to ~15%; i.e., it becamenearly fully saturated. As expected, fractional reabsorption during EDmicroinfusion 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, thesignificance level was just reached ( P 0.05). Thesedata demonstrate that myo -[ 3 H]inositolreabsorption from the loop of Henle is mediated by a high-capacitytransporter. Apparent kinetic parameters roughly estimated from the LPvalues of Fig. 2 were as follows: J max = 0.88 ± 0.14 pmol · s 1 · shortloop of Henle 1; K m (concentrationat 1/2 J max ) = 3.4 ± 1.67 mmol/l ( n = 36).9 e: L0 d- u% p; w* m0 v0 w, }- v

When we microinfused a solution containing 90 µmol/l 3 H-labeled myo -inositol plus[ 14 C]inulin into the ascending limb of an LLH near thehairpin bend, 44 ± 4.6% ( n = 15) of myo -[ 3 H]inositol was reabsorbed between thepuncture site and the urine emerging from the ipsilateral ducts ofBellini. This fraction did not change when 0.1 mmol/l phloridzin or 50 mmol/l nonlabeled myo -inositol was added to themicroinfusate (see Fig. 3 ). Thus myo -inositol is able toleave the lumen of tubule segments that are situated downstream fromthe LLH microinfusion site. Because ED microinfusion of myo -[ 3 H]inositol did not result in anyreabsorption (see Fig. 2 ), the collecting duct is not involved in thereabsorptive process. Thus the myo -[ 3 H]inositol microinfused into LLH musthave been reabsorbed in the ascending limb of Henle's loop ofjuxtamedullary nephrons.

Microinfusing the same solution (90 µmol/l myo -[ 3 H]inositol plus[ 14 C]inulin) into vasa recta running parallel to the LLH,we found that fractional recovery was 27 ± 0.05%( n = 8) in the ipsilateral urine and 1.8 ± 0.007% ( n = 7) in the contralateral urine. The formervalue decreased significantly to 12 ± 0.02% ( n = 7) (and the contralateral one did not change) when 0.1 mmol/lphloridzin or 50 mmol/l nonlabeled myo -inositol was added tothe microinfusate (see Fig. 4 ). Thus myo -[ 3 H]inositol is also able to enter thetubular urine from the ipsilateral vasa recta blood in aphloridzin-sensitive and saturable manner.) j, H7 K, G: C# o

Localization, 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 theloop of Henle (see below), we first had to characterize D -glucose reabsorption in experiments similar to those usedwith myo -inositol. First, we determined the fractionalreabsorption of 10 µmol/l 3 H-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 reflectsnearly exclusively reabsorption in short loops of Henle, becausereabsorption 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 amountedto 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. Inthe presence of 1 mmol/l phloridzin, the fractional reabsorption of 10 µmol/l D -[ 3 H]glucose decreased to 9.5 ± 1.6 (EP, n = 6) and to 12.5 ± 2.4% (LP, n = 7; see Fig. 7 ). Therefore, D -glucosereabsorption in short loops of Henle was not only saturable but alsosensitive to phloridzin.
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When we microinfused a solution containing 33 µmol/l 3 H-labeled D -glucose plus[ 14 C]inulin into the ascending limb of an LLH near thehairpin bend, 31 ± 8% ( n = 10) of D -[ 3 H]glucose was reabsorbed between thepuncture site and the urine emerging from the ipsilateral ducts ofBellini. This fraction did not change significantly when 0.1 mmol/lphloridzin or 50 mmol/l nonlabeled D -glucose was added tothe microinfusate (see Fig. 3 ). Thus D -glucose is able toleave the lumen of tubule segments that are situated downstream fromthe LLH microinfusion site. As ED microinfusion of D -[ 3 H]glucose resulted only in a very smallfractional reabsorption, the collecting duct does not seem to beinvolved in the reabsorptive process. Thus the D -[ 3 H]glucose microinfused into LLH must havebeen reabsorbed in the ascending limb of Henle's loop ofjuxtamedullary nephrons.

Microinfusing the same solution (33 µmol/l D -[ 3 H]glucose plus[ 14 C]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 significantlywhen 0.1 mmol/l phloridzin or 50 mmol/l nonlabeled D -glucose was added to the microinfusate. Thus D -[ 3 H]glucose is also able to enter thetubular urine from the ipsilateral vasa recta blood.) `4 C4 b' a7 i# u: p8 F2 W
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Molecular specificity of myo-inositol vs. D -glucosereabsorption in the loop of Henle. In further sets of experiments, we evaluated the molecular specificityof the carrier(s) involved in tubular myo -[ 3 H]inositol reabsorption during LPmicroinfusion. For this purpose, 50 mmol/l of the following compoundswere added to the microinfusate containing 10 µmol/l 3 H-labeled myo -inositol plus[ 14 C]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 largeinhibitory effect as nonlabeled myo -inositol itself, whereas L - chiro -inositol, D -glucose, D -galactose, and -methyl- D -glucoside had amuch smaller but significant inhibitory effect on myo -[ 3 H]inositol reabsorption. No inhibitionoccurred in the presence of the remaining four compounds.

Fig. 5. Molecular structure of inositol stereoisomers used inthis report and D -glucose.
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Fig. 6. Fractional 3 H reabsorption [=100 fractional recovery of 3 H in the final urine (in %)]during microinfusion (10 nl/min) of 10 µmol/l myo -[ 3 H]inositol into LP segments ofsuperficial nephrons. The individual compounds (50 mmol/l, except forphloridzin) were added ( ) to the microinfusion solution. Values aremeans ± SE; the no. of microinfused nephron segments is inparentheses.
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The small effect of D -glucose on the reabsorption of myo -inositol (Fig. 6 ) could have meant that myo -inositol is transported by one of the D -glucose transporters (SGLT1 and/or 2) at a significantly higher affinity than D -glucose itself. Therefore, we testedthe extent to which myo -inositol and the strong inhibitor ofits reabsorption, D - chiro -inositol (see Fig. 6 ),as well as other sugars and polyols influenced reabsorption of D -glucose. For this purpose, we microinfused 10 µmol/l D -[ 3 H]glucose (plus[ 14 C]inulin) at LP microinjection sites in the presenceof 50 mmol/l of nonlabeled myo -inositol, D - chiro -inositol, L - chiro -inositol, D -fructose, L -fucose, 3- O -methyl-glucose, D -galactose, and -methyl- D -glucoside. The results are depictedin Fig. 7. Whereas the reabsorption of D -glucose was 91.3% at a concentration of 10 µmol/l(control) and was reduced to 35.5% at 50 mmol/l, it was inhibited toonly a moderate or small extent by the addition of 50 mmol/l ofnonlabeled D -fructose, -methyl- D -glucoside,or D -galactose. Fractional reabsorption of D -glucose in the presence of these three compounds was62.8 ± 9.4, 66.4 ± 4.9, and 81.9 ± 5.6%,respectively. At 50 mmol/l myo -inositol,3- O -methyl-glucose(=3- O -methyl- D -glucopyranose), D - chiro -inositol, L - chiro -inositol, and L -fucose(=6-deoxy- L -galactose) did not have any significant effecton D -[ 3 H]glucose reabsorption (Fig. 7 ).7 w7 T/ h( [# [+ p3 ?$ l+ @) r

Fig. 7. Fractional 3 H reabsorption [=100 fractional recovery of 3 H in the final urine (in %)]during microinfusion (10 nl/min) of 10 µmol/l D -[ 3 H]glucose into LP segments of superficialnephrons. The individual compounds (50 mmol/l, except for phloridzin)were added ( ) to the microinfusion solution. Values are means ± SE; the no. of microinfused nephron segments is in parentheses.- r2 Y( h6 B. p; Y# q* k
6 v1 a+ m7 r" i+ N' V" g& r' ?( o
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/lwas higher during EP than during LP microinfusion. This means that thetubule segment located between these two microinfusion sites, i.e., the PCT, contributes to renal myo -inositol reabsorption. Tostudy this process in more detail directly, we microperfused thesegment between EP and LP micropuncture sites with a solutioncontaining 10 µmol/l 3 H-labeled myo -inositol(  [ 14 C]inulin) in the absence and presence of 0.1 mmol/lphloridzin or 50 mmol/l nonlabeled myo- inositol, D -glucose, L -fucose, and 3- O -methylglucose at a microperfusion rate of 20 nl/min. Thefractional recovery of the 3 H activity was determined inthe 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 decreasedto 9.2 ± 2.3, 36.3 ± 2.2, or 17.3 ± 2.4% in thepresence 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 asignificant effect. Qualitatively, these results resemble those for theshort loops of Henle (see Fig. 6 ), but fractional reabsorption wasgenerally lower in the microperfused segment of the proximalconvolution than that in short loops of Henle. The microperfusionsolution had a higher pH than that of the LP microinfusion experiments (see MATERIALS AND METHODS ). However, thiscannot be the reason for the quantitative difference in reabsorption,because myo- inositol reabsorption was pH independent in thispH range.
' i+ n$ S* \* y% u& j1 u
Fig. 8. Fractional 3 H reabsorption [=100 LPfractional recovery of 3 H (in %)] during microperfusion(20 nl/min) of 10 µmol/l myo -[ 3 H]inositolbetween EP to middle proximal and LP micropuncture sites of superficialnephrons. The individual compounds (50 mmol/l, except for phloridzin)were added ( ) to the microperfusion solution. Values are means ± SE; the no. of microperfused nephron segments is in parentheses.

Reabsorption of D -glucose in the PCT. In additional EP microinfusion experiments, we found that fractionalreabsorption of D -glucose at an initial concentration of 50 mmol/l (70.1 ± 4.0%, n = 5) was much higher thanduring the LP microinfusion reported above (35.5 ± 4.8%, n = 5). This last finding was not unexpected andindicates 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 thesegment between EP and LP micropuncture sites with a solutioncontaining 10 µmol/l 3 H-labeled D -glucose (  14 C-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, fractionalreabsorption of 10 µmol/l D -glucose was 74.1 ± 2.8% (control) and decreased to 15.7 ± 2.5% in the presence of50 mmol/l nonlabeled D -glucose and to 16.5 ± 1.4 and8.0 ± 3.3.% in the presence of 0.1 and 1 mmol/l phloridzin,respectively. L -Fucose and 3- O -methylglucose hada small effect, whereas 50 mmol/l myo -inositol did notinfluence D -glucose reabsorption at all. Qualitatively,these results again mirror those from the short loops of Henle (seeFig. 7 ), but fractional reabsorption is generally lower in themicroperfused segment of the proximal convolution than in the shortloops of Henle., K$ p% L+ [. i, ?, n
8 Q$ ?/ L; A1 C1 O
Fig. 9. Fractional 3 H reabsorption [=100 LPfractional recovery of 3 H (in %)] during microperfusion(20 nl/min) of 10 µmol/l D -[ 3 H]glucosebetween EP to middle proximal and LP micropuncture sites of superficialnephrons. The individual compounds (50 mmol/l, except for phloridzin)were added ( ) to the microperfusion solution. Values are means ± SE; the no. of microperfused nephron segments is in parentheses.& a" Q7 Y$ v( k& p3 ^

DISCUSSION; R) P0 R% d: b/ ?9 X

It has been known for about 50 years that myo -inositolis nearly completely reabsorbed in the mammalian kidney( 6 ). However, only very little has been known about thelocalization of the reabsorptive process along the nephron and thetransport mechanism and its molecular specifity. We investigated thesetopics by microinfusing and microperfusing single tubule segments ofrat kidney in vivo 60% of thephysiological glomerular load of myo -inositol can bereabsorbed in the PCT. 90% of a myo -inositol load that is higherthan the physiological glomerular load. In both segments, myo -inositol reabsorption was phloridzin sensitive andsaturable. These findings are in accord with earlier in vitro resultsshowing that renal cortical brush-border membrane vesicles (BBMV) fromrat and rabbit take up myo -inositol by a saturable,phloridzin-sensitive process that is stimulated by an electrochemicalNa gradient ( 11, 27 ).2 t; W; x6 W5 R: ^
$ ~# s# W5 q( d
Essentially, no myo -inositol reabsorption occurred innephron segments beyond the short loops of Henle and in the collecting ducts. This means that reabsorptive data obtained during LPmicroinfusions represent reabsorption in short loops of Henle. Our dataobtained by microinfusing the ascending limb of LLH revealed that about one-half of the microinfused myo -[ 3 H]inositolwas reabsorbed downstream from this micropuncture site. Thereabsorption must take place in the ascending limb of LLH because thecollecting ducts are not able to reabsorb myo -inositol (seeabove). Moreover, myo -[ 3 H]inositolmicroinfused into inner medullary AVR appeared in the ipsilateral finalurine to a much greater extent than in the contralateral urine. Thistransport out of the vasa recta was saturable and sensitive tophloridzin. 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 theconnecting tubule in the medullary ray, but we do not think that it is likely.
. y, F$ S( b$ L8 M  m: f
In these studies, we have shown that myo -inositolreabsorption in the short loops of Henle and in the PCT is nearlycompletely saturated if a high concentration of nonlabeled myo -inositol is added to the microinfusate. To test thespecificity of this transport and, at the same time, to get an idea ofwhich apical carrier(s) is involved in tubular myo -inositolreabsorption, we tried to inhibit it with high concentrations ofseveral polyols and sugars. We observed a strong inhibition of myo -inositol reabsorption when it was infused together withthe myo -inositol derivatives scyllo -inositol (orscyllitol) or D - chiro -inositol. Scyllo -inositol has previously been shown to inhibitstrongly myo -inositol uptake into rat BBMV ( 27 ). Moreover, scyllo -inositol induces nearlythe same steady-state current as myo -inositol in oocytesinto which cRNA of canine SMIT has been injected ( 10 ).8 {0 @- i4 @. G3 j9 k
) r3 W  F* N- m& V0 `) X
As illustrated in Fig. 6, D -fructose, D -mannose, L -fucose, and3- 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 GLUT5or a mannose symporter ( 7, 24 ).* g* H8 ]+ i& j9 \9 L
, a- ?; M# o9 H+ i: l. c
We tested L -fucose and 3- O -methylglucose becauseprevious studies have shown that uptake currents in canineSMIT-injected oocytes are high with L -fucose but very lowwith 3- O -methylglucose ( 10 ). In the same paper,uptake current was also measured in oocytes transfected with rabbitNa -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 -inositoltransport in the short loop of Henle (Fig. 6 ) or the PCT (Fig. 8 ),could mean that myo -inositol is not reabsorbed by eitherSMIT or SGLT1. However, although SMIT from the dog and SMIT from therat have a 95% homology at the protein level (358 amino acids inboth), dog cDNA (2,870 bases) is much longer than rat cDNA (1,155 bases) ( 15, 17, 35 ). Therefore, the specificity of canineSMIT might be quite different from that of the rat because splicevariants of SMIT have been found even in the same species ( 8, 22 ). Thus we cannot rule out the possibility that SMIT isresponsible for the reabsorption of myo -inositol in the rat kidney." Y, ?5 E! e4 T

However, are the D -glucose transporters in tubule apicalmembranes involved in myo -inositol reabsorption? Tubular D -glucose reabsorption shows axial heterogeneity( 29 ). SGLT1 is present in the S3 segment and SGLT2, in theS1 and S2 segments of the proximal tubule ( 14, 16 ).Substrates of the rat and rabbit SGLT1 are D -glucose, -methyl- D -glucoside, D -galactose, and3- O -methyl- D -glucose ( 10, 16 );substrates of the rabbit and pig SGLT2 are D -glucose and -methyl- D -glucoside, but not D -galactose and3- O -methyl- D -glucose ( 14, 19 ).However, localization and/or specificity of these transporters does notseem to be clear cut because D -galactose is reabsorbed inthe PCT ( 32 ), which does not include the S3 segment. Inthe present study, D -glucose, -methyl- D -glucoside, and D -galactose, butnot 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 -inositolis reabsorbed by the SGLT carriers. However, D -glucose uptake currents in oocytes transfected with canine SMIT are small andnot different for D - and L -glucose. Thisobservation hardly seems compatible with the hypothesis that SGLTsrepresent the apical myo -inositol transporters in the kidneybecause glucose transport by SGLTs is highly stereospecific.6 K1 l5 S* `/ c: g6 S- H1 E9 g

To further test the hypothesis that myo -inositol isreabsorbed 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 -inositolreabsorption. In both segments, marked D -glucosereabsorption, which was phloridzin sensitive and saturable, took place.The roughly estimated kinetic constants were similar to thosedetermined 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 J max and K m for D -glucose reabsorption in the short loops of Henle areabout fourfold higher than those for myo -inositol reabsorption.. m( |4 B, _  a; J0 Q( @

D -Glucose reabsorption was inhibited to a moderate extentby -methyl- D -glucoside in the short loop of Henle and by3- O -methyl- D -glucose in the PCT. However, in thecontext of this paper, it is most important that neither myo- inositol nor D - chiro -inositolinfluenced D -glucose reabsorption in either segment to anyextent. These results clearly show that the SGLT carriers do not accept myo -inositol to a significant extent, thereby confirmingearlier results that showed that myo -inositol does not haveany influence on radiolabeled -methyl- D -glucoside uptakein SGLT1- ( 16 ) or SGLT2-transfected oocytes( 14 ).
/ q- z) U9 [5 G3 S  y4 P) N
A further possibility is that tubular reabsorption of myo -inositol is mediated by theH - myo -inositol symporter (HMIT) expressedpredominantly in the mammalian brain ( 31 ) but apparentlyto a small extent also in the kidney. Increasing the driving force forH uptake into oocytes by decreasing the extracellular pHfrom 7 to 5 increased myo- inositol transport roughly sixfold( 31 ). However, in the present work, increasing the pH ofthe microinfusate from 6.7. to 7.6 did not have any effect on myo -inositol reabsorption in the short loops of Henle. Thusit seems to be unlikely that the tubular reabsorption investigated inthis paper is mediated by HMIT. The fact that the uptake of myo -inositol into tubular BBMV is driven by aNa gradient ( 10, 25 ) also speaks against thispossibility and also against tubular reabsorption of myo -inositol by a uniporter like GLUT5.0 }6 ?. ?% I' t: k3 i

Most recently, an orphan cDNA 43% identical in sequence to SMIT[now called SMIT1 ( 3 )] was expressed in oocytes thatwere subsequently voltage clamped ( 3 ). Inward currentswere found during superfusion with myo- inositol, D - chiro -inositol, and, to a smaller extent, with D -glucose. Uptake by this transporter (called SMIT2 by theauthors) exhibited stereospecificity for D -glucose and D - chiro -inositol. L -Fucose was notaccepted by SMIT2. This specificity resembles not only that foundearlier when myo -inositol uptake was studied in liver cells( 21 ) but also that of our present data. Thus wehypothesize that SMIT2 is responsible for renal tubular reabsorption of myo -inositol./ s7 r" E  E! F3 P! Q: C

We conclude from our data that tubular reabsorption of myo -inositol in the PCT and in the loop of Henle isresponsible for the nearly complete fractional reabsorption of thiscompound. Myo -inositol reabsorption is not mediated by theSGLTs, the HMIT carrier, the mannose transporter, or the GLUT5uniporter. Our data support our hypothesis that myo -inositolreabsorption across the luminal membrane of the PCT, the short loop ofHenle, and the ascending limb of the LLH is mediated by SMIT2. Theextent to which this luminal route is used for the high myo -inositol accumulation in the cells of the thickascending limb of Henle's loop ( 23 ) and for renalintracellular inositol metabolism remains to be elucidated." d& J# _# v+ |& D7 s- t, w

ACKNOWLEDGEMENTS
4 H! ]( h0 M6 m0 R) j
We thank Olga Brokl and Kristen Evans for encouragement and supportin the laboratory at the University of Arizona and Dr. S. H.Wrightfor valuable discussions.3 ~+ y& h+ N" v) O' w
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