Exploring the role of galectin 3 in kidney function: a genetic approach

Glycobiology vol. 16 no. 1 pp. 36–45, 2006 doi:10.1093/glycob/cwj035 Advance Access publication on September 15, 2005

© The Author 2005. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: [email protected] 36

Exploring the role of galectin 3 in kidney function: a genetic approach

Maurice Bichara2,3,4, Amel Attmane-Elakeb2,3,4, Dennis Brown5, Marie Essig6,4, Zoubida Karim2,3,4, Martine Muffat-Joly7, Laetitia Micheli8, Isabelle Eude-Le Parco9, Françoise Cluzeaud3,4,10, Michel Peuchmaur11, Jean-Pierre Bonvalet3,4,10, Françoise Poirier1,9, and Nicolette Farman3,4,10

2INSERM U426, 16 rue Henri Huchard, 75870 Paris Cedex 18, France; 3IFR 2 Claude Bernard, 16 rue Henri Huchard, 75870 Paris Cedex 18, France; 4Université Paris 7, 2 place Jussieu, 75005 Paris, France; 5Program in Membrane Biology/Renal Unit, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114; 6Service de Néphrologie, Hôpital Xavier Bichat, 46 rue Henri Huchard, 75870 Paris Cedex 18, France; 7Centre d’Explorations Fonctionnelles Intégrées, IFR 2 Claude Bernard, 16 rue Henri Huchard, 75870 Paris Cedex 18, France; 8Association Claude Bernard, Centre de recherche de génétique et pathologie moléculaire de l’hématopoièse, 16 rue Henri Huchard, 75870 Paris Cedex 18, France; 9Institut Jacques Monod, CNRS UMR 7592, Universités Paris 6 and Paris 7, 2 place Jussieu, 75251 Paris Cedex 05, France; 10INSERM U478, 16 rue Henri Huchard, 75870 Paris Cedex 18, France; and 11Equipe EA 3102, Service d’Anatomopathologie, Hôpital Robert Debré, 75019 Paris, France

Received on May 30, 2005; revised on August 31, 2005; accepted on September 1, 2005

Galectin 3 belongs to a family of glycoconjugate-binding pro-teins that participate in cellular homeostasis by modulatingcell growth, adhesion, and signaling. We studied adult galec-tin 3 null mutant (Gal 3–/–) and wild-type (WT) mice to gaininsights into the role of galectin 3 in the kidney. By immuno-fluorescence, galectin 3 was found in collecting duct (CD)principal and intercalated cells in some regions of the kidney,as well as in the thick ascending limbs at lower levels. Com-pared to WT mice, Gal 3–/– mice had ~11% fewer glomeruli(p < 0.04), associated with kidney hypertrophy (p < 0.006). Inclearance experiments, urinary chloride excretion was foundto be higher in Gal 3–/– than in WT mice (p < 0.04), but therewas no difference in urinary bicarbonate excretion, in glomer-ular filtration, or urinary flow rates. Under chronic lowsodium diet, Gal 3–/– mice had lower extracellular fluid(ECF) volume than WT mice (p < 0.05). Plasma aldosteroneconcentration was higher in Gal 3–/– than in WT mice (p <0.04), which probably caused the observed increase in �-epi-thelial sodium channel (�-ENaC) protein abundance in themutant mice (p < 0.001). Chronic high sodium diet resultedparadoxically in lower blood pressure (p < 0.01) in Gal 3–/–than in WT. We conclude that Gal 3–/– mice have mild renalchloride loss, which causes chronic ECF volume contractionand reduced blood pressure levels.

Key words: blood pressure/body fluid volumes/hyperfiltra-tion/Bartter’s like syndrome/null mutant mouse

Introduction

Lectins are proteins specifically binding to glycoconjugates.Lectins play important roles in several normal and patho-logical processes through largely unknown molecularmechanisms. Among lectins, galectins are specific for beta-galactoside derivatives and share a unique structure that isentirely different from other types of lectins (Barondes et al.,1994a,b). The mammalian galectin gene family includes 12members (Houzelstein et al., 2004). Galectins are relativelysmall proteins (14–30 kDa), and, despite the lack of signalpeptide, they can be found both inside and outside the cellsdepending on the cell type and on the stage of differentia-tion (Barondes et al., 1994a,b). This latter unusual propertymay at least partly account for the broad spectrum ofpotential functions that have been ascribed to galectins.Hence, there is a large body of evidence showing that galec-tins could act not only as modulators of cell–cell or cell–substratum interactions but also as apoptotic (or antiapop-totic) molecules (Hughes, 2001; Liu et al., 2002). Galectin 3is normally expressed in the developing human (Winyardet al., 1997) and mouse (Bullock et al., 2001) kidney whereit has been proposed to play a role in fetal collecting duct(CD) morphogenesis since exogenous addition of galectin 3to embryonic mouse kidney explants was shown to inhibitbranching of the ureteric bud (Bullock et al., 2001). Morerecently, it has been proposed that galectin 3 participates inthe functional adaptation to metabolic acidosis of interca-lated cells of the renal CD (Hikita et al., 2000). The A- andB-intercalated cells in the collecting tubules of the kidneyare responsible for the final acidification or alkalinizationof the urine. Metabolic acidosis is proposed to remodelB-intercalated (bicarbonate-secreting) cells into A-intercalated(acid-secreting) cells (Al-Awqati, 2003). A similar remodel-ing of cell polarity has been characterized in detail in animmortalized cell line, and this process requires the deposi-tion of hensin in the extracellular matrix. Hensin is a 230kDa extracellular glycoprotein that can induce, in its poly-merized form, terminal differentiation of renal intercalatedcells cultured in vitro (Hikita et al., 2000). The signal for ter-minal differentiation of intercalated cells appears to be thesecretion of galectin 3 (Hikita et al., 2000). Once outside thecells, galectin 3 binds to and promotes polymerization ofhensin, and the hensin-galectin 3 complex is able to inducethe conversion of B-like intercalated cells into A-like inter-calated cells in vitro (Hikita et al., 2000). Little is knownabout the localization and the role of galectin 3 in the kid-ney in vivo. In humans, galectin 3 expression decreases after

1To whom correspondence should be addressed; e-mail: [email protected]

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birth and was noted to be restricted to a subset of cells inthe CD in infants (Winyard et al., 1997). Renal expressionof galectin 3 mRNA and protein was shown to be upregu-lated in acute renal failure in the rat, thus suggesting a rolein acute tubular injury and the subsequent regenerationstage (Nishiyama et al., 2000). In the kidney of normal rat,a very weak galectin 3 signal was found to be confined todistal tubules (Nishiyama et al., 2000). However, the precisecell type was not identified with established markers inthese experiments.

To assess the role of galectin 3 in vivo, we have studiedGal 3 null mutant mice (Gal 3–/–) (Colnot et al., 1998a).These Gal 3–/– mice are viable and fertile but severaldefects associated with this mutation have been observedand indicate that galectin 3 plays a role in inflammation(Colnot et al., 1998b) and in endochondral ossification(Colnot et al., 2001). It has also been shown that theabsence of galectin 3 leads to an accelerated glomerulopa-thy after streptozotocin-induction of diabetes, which wasattributed to disruption of the capacity of galectin 3 to bindtoxic advanced glycation end products (Pugliese et al.,2001). The aim of this study was to search for a function ofgalectin 3 in the kidney glomerular and tubular functions inadult mice. We found that Gal 3–/– mice have a reducednumber of nephrons without renal insufficiency, and thatthey show an increased renal ability to excrete chlorideleading to extracellular fluid (ECF) volume contraction andlower blood pressure after challenge with high salt diet ascompared to wild-type (WT) mice.

Results

Kidney histology and immunolocalization of Galectin 3

By standard histological examination, the kidneys of Gal3–/– mice seemed normal and did not appear to be differentfrom those of WT mice (data not shown). Cell-specificexpression of galectin 3 on kidney sections is illustrated inFigure 1. In the cortex of WT mice, a minority of tubulesappeared to express galectin 3 and, as expected, no signalwas present over sections of Gal 3–/– mice (data notshown). In the cortical CD (Figure 1A and C), a majority ofcells had positive galectin 3 signal, suggesting predominantexpression in principal cells (PCs) that represent ~60% ofthe cells in this tubular segment in the mouse (Kim et al.,1999). This is confirmed by colocalization of galectin 3 andaquaporin 2 (AQP 2), a marker of PCs (Figure 1A, panelb). Staining with anti-proton pump (PP) antibodiesrevealed that most B-type intercalated cells in the cortexalso contained galectin 3, but the staining intensity waslower than in PCs (Figure 1C, cortex). In contrast, many A-type intercalated cells in the cortex were not stained, or onlyweakly-stained for galectin 3, although an occasional A-cellwas more brightly stained. A similar pattern was seen in theouter stripe of the outer medulla, where PCs were positiveand most A-intercalated cells were unstained (Figure 1C,outer stripe). This pattern of staining in the cortical and outermedullary CDs was confirmed by immuno-electron micros-copy (Figure 2) which revealed unstained A-intercalated cellsadjacent to heavily-stained PCs in cortical and outer med-ullary (outer stripe) CDs. Cortical B-intercalated cells were

moderately stained (Figure 2). In contrast, both A-interca-lated cells and PCs in the inner stripe and in the innermedulla were equally well-stained for galectin 3 (Figure 1C,inner stripe). Galectin-3 positive cells were also present intubules that express uromucoid (Figure 1A, panel c), indi-cating that galectin 3 is also expressed in the thick ascend-ing limb of Henle’s loop (TAL). In contrast, proximaltubules and glomeruli were consistently negative. Regionsof transition between the S3 segment of the proximal tubuleand the thin descending limb of Henle’s loop (TDL) ofHenle showed that TDLs were also positive for galectin 3(Figure 1B). These results indicate that galectin 3 in adultmouse kidney is expressed in CD PCs and B-intercalatedcells, as well as A-intercalated cells in the inner stripe andthe inner medulla. Thick ascending limbs of Henle are alsopositive. Galectin 3 seemed mainly localized inside the cells(both cytoplasm and nucleus in some cases) without appar-ent extracellular staining.

Renal function study

In 15- to 31-week-old mice fed with a standard sodium diet,there was no difference in hematocrit nor in plasma valuesof protein, sodium, potassium, chloride, and bicarbonatebetween WT and Gal 3–/– mice (Table I). Clearance experi-ments were performed under two different conditions. First,mice were perfused with isotonic saline. As summarized inTable II, the glomerular filtration and urinary flow ratesand the sodium and potassium excretion rates were compa-rable in the two strains of mice. However, Gal 3–/– mice hada significantly higher (p < 0.04) chloride excretion rate thanWT mice (Tables I–III). Note that there was no significantdifference in the filtered loads of chloride between WT(47,294 ± 7652 nmol/min) and Gal 3–/– (48,011 ± 5784 nmol/min) mice. Since the sum of sodium plus potassium minuschloride urinary concentration was the same in both groupsof mice (64 ± 15 in WT vs. 51 ± 16 mM in Gal 3–/–, n = 6 ineach group, NS), it appears that Gal 3–/– mice excretedmore NaCl plus KCl than WT mice. Second, mice were per-fused with 150 mM NaHCO3 to measure urinary bicarbon-ate excretion rates with accuracy. WT and Gal 3–/– micehad the same degree of metabolic alkalosis at the end of theclearance experiments (Table III). There was no differencein the glomerular filtration rate (GFR), urinary flow rate,and bicarbonate excretion rate between WT and Gal 3–/–mice (Table III). Of interest Gal 3–/– mice had a signifi-cantly lower mean arterial pressure than WT in this experi-mental series (p < 0.003; Table III).

The number of glomeruli per kidney was ~11% lower inGal 3–/– than in WT mice (p < 0.04; Table IV), but thekidney weight was higher in Gal 3–/– than in WT mice(p < 0.006; Table IV). These results indicate that there wasa lower number of nephrons with compensatory kidneyhypertrophy in Gal 3–/– mice. However, this did not leadto chronic renal insufficiency in aging Gal 3–/– mice, asestimated by measurements of plasma creatinine (38 ± 1vs. 40 ± 2 μM; NS) and urea (10.2 ± 1.2 vs. 11.8 ± 0.8 mM;NS) in 1-year-old WT and Gal 3–/– mice. Plasma phos-phate (1.9 ± 0.3 vs. 2. 6 ± 0.2 mM) and glucose (8 ± 1 vs.10 ± 2 mM) concentrations were also comparable in WTand Gal 3–/– old animals.

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Fig. 1. Immunofluorescence studies of galectin 3 expression in kidney. Kidney sections of WT mice were immunostained with anti-galectin 3 antibody (in red) alone or in the presence (in green) of cell markers. Panel A: A minority of tubules were positive for galectin 3 in the cortex (a and b) and medulla (c). A majority of CCD cells, presumably corresponding to PCs, were clearly galectin 3-positive (a, arrows), whereas some cells were weakly or not stained (a, arrowheads), presumably the intercalated cells. Colocalization of galectin 3 and AQP2 fluorescence (in yellow) was clear in the cortical CDs (b). Galectin 3 was also found in the thick ascending limb of Henle’s loop, colocalized with uromucoid (in yellow, c). mTAL, medullary portion of the thick ascending limb of Henle’s loop. Bars: in panel a, 20 μm, and 50 μm in b and c. Panel B: Frontier between the outer stripe and the inner stripe of the outer medulla showing a transition between a proximal tubule PT S3 segment and a TDL. The proximal tubule is unstained for Gal 3, whereas the thin descending limb contains Gal 3. TALs show a moderate Gal 3 staining. Bar, 30 μm. Panel C: Cortex, localization of Gal 3 and the vacuolar ATPase PP (56 kD B1-subunit) in the cortex. CDs are Gal 3-positive, whereas surrounding proximal tubules are unstained. PP-negative PCs are Gal 3-stained and B-intercalated cells identified by their basolateral staining are also Gal 3-stained (A and B, inset). In contrast, A-intercalated cells with apical PP staining were mostly negative for Gal 3 (A, inset). Bar, 20 μm. Outer stripe: Localization of Gal 3 and PPs in CDs from the outer stripe of the outer medulla. In this tubule segment, PCs are stained for Gal 3, whereas A-intercalated cells (identified by positive PP staining, arrows) are negative for Gal 3. Bar, 30 μm. Inner stripe: Localization of Gal 3 and PPs in the inner stripe of the outer medulla. In this segment, all CD epithelial cells are positive and the intensity of Gal 3 staining is variable among both the PCs and the PP positive intercalated cells population. The papillary surface epithelium is Gal 3 positive (top right of image). Bar, 30 μm.

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Systolic blood pressure in conscious mice

In these series, WT and Gal 3–/– mice were maintainedunder low (0.038% Na) or high (3% Na) sodium intake fora period of 5 months after weaning and noninvasive tail-cuff blood pressure was measured each month in consciousanimals as described in the Materials and methods section.Although there was no difference in the blood pressure values

as a function of time within any group of mice, the valuesobtained in each group during the study were pooled. Asshown in Figure 3, there was no significant difference in theblood pressure values between Gal 3–/– and WT mice sub-mitted to the low sodium diet. In contrast, chronic highsodium diet allowed to reveal a striking and unexpecteddifference, because systolic blood pressure levels were then

Fig. 2. Immuno-electron microscopy. Panel A: Immunogold electron microscopy of a CCD showing cytoplasmic labeling for Gal 3 in a PC. An adjacent A-intercalated (A-IC) cell, identified by its characteristic appearance (apical microvilli and many cytoplasmic vesicles), is negative. Bar, 0.5 μm. Panel B: Immunogold electron microscopy of a CCD showing a labeled PC and an adjacent B-intercalated (B-IC) cell which shows moderate amount of immunogold labeling. The B-cell contains more mitochondria than the PC but has fewer apical microvilli than the A-IC cell shown in the previous figure (Figure 2A). Bar, 0.5 μm. Panel C: Immunogold electron microscopy of a CD from the outer stripe of the outer medulla showing cytoplasmic labeling for Gal 3 in a PC. An adjacent A-intercalated (A-IC) cell, identified by its characteristic appearance (apical microvilli and many cytoplasmic vesicles), is negative. Bar, 0.5 μm.

Table I. Assessment of renal function in WT and Gal 3–/– mice: arterial values

Gal 3–/–, galectin 3 null mutant mice; N, number of mice; NS, not significant; WT, wild type.The data represent mean ± SEM.

Hematocrit (%) Protein (g/L) [Na+] (mM) [K+] (mM) [Cl–] (mM) [HCO3–] (mM)

WT (N = 6) 46.2 ± 0.7 58 ± 3 150 ± 2 4.5 ± 0.3 125 ± 2 22 ± 1

Gal 3–/– (N = 5) 44.5 ± 1.2 57 ± 5 153 ± 2 5.3 ± 0.6 125 ± 2 24 ± 1

NS NS NS NS NS NS

Table II. Assessment of renal function in WT and Gal 3 –/– mice: clearance study in mice perfused with isotonic saline

Gal 3–/–, galectin 3 null mutant mice; GFR, glomerular filtration rate; MAP, mean arterial pressure; N, number of mice; NS, not significant; WT, wild type.The data represent mean ± SEM.

MAP (mmHg) GFR (μL/min)Urinary flowrate (μL/min)

Na excretion rate (nmol/min)

K excretion rate (nmol/min)

Cl excretion rate (nmol/min)

WT (N = 6) 103 ± 6 342 ± 55 1.7 ± 0.2 257 ± 54 153 ± 10 321 ± 35

Gal 3–/– (N = 6) 90 ± 7 356 ± 42 1.8 ± 0.2 331 ± 76 230 ± 60 463 ± 61

NS NS NS NS NS p < 0.04

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significantly lower in Gal 3–/– (141 ± 3 mmHg, N = 9 ani-mals) than in WT (156 ± 5 mmHg, N = 4 animals; p < 0.01)mice (Figure 3).

Body fluid volumes

Body weight and body fluid volumes were assessed in 29-week-old WT and Gal 3–/– mice maintained on low or highsodium diet for 6 months. In the low sodium diet conditions,Gal 3–/– animals had significantly lower body weight (24.1 ±0.8 vs. 29.8 ± 0.3 g in WT; p < 0.0001), less total body water(TBW) (24.3 ± 0.9 arbitrary units vs. 31.2 ± 3.7 in WT; p <0.05), and less extracellular water (ECW) (8.3 ± 0.4 arbitraryunits vs. 9.8 ± 0.6 in WT; p < 0.04) than age-matched WTmice. Intracellular water (ICW) content did not differbetween Gal 3–/– and WT mice. In the high sodium dietconditions, there was a high mortality rate at 7 months ofage in the WT group so that only two WT mice could bestudied (body weight: 28.2 and 34.0 g). In contrast, Gal 3–/–mice did not die and eight mice could be examined (bodyweight, 28.0 ± 0.4 g; TBW, 27.9 ± 0.8 arbitrary units; ECW,9.2 ± 0.1 arbitrary units; ICW, 18.8 ± 0.7 arbitrary units).

Plasma aldosterone concentration

Plasma aldosterone concentrations were measured in micewith normal diet or after 6 months on a low or high sodiumdiet. Plasma aldosterone concentrations were significantlyhigher in Gal 3–/– than in WT mice maintained on both thelow (1026 ± 63 in Gal 3–/– vs. 659 ± 132 pg/mL in WT; p <0.01) and normal (158 ± 33 in Gal 3–/– vs. 80 ± 18 pg/mL inWT; p < 0.04) sodium diet. For mice maintained on a highsodium diet, only two values of plasma aldosterone concen-tration (64 and 52 pg/mL) could be obtained in WT micebecause of the high mortality rate as mentioned above. Bycontrast, nine values were obtained in Gal 3–/– mice (97 ±22 pg/mL). Altogether elevated plasma aldosterone levels inGal 3–/– mice are consistent with the renal salt wasting andECF volume contraction described above in these animals.

Quantification of transport proteins

Since Gal 3–/– mice excreted more NaCl plus KCl than WTmice, the abundance of some transport proteins expressedin the CDs was assessed by western blot experiments inmice subjected to the standard diet. In the renal cortex,Figure 4 shows that there was no difference in the abun-dance of the Na/K-ATPase α-subunit nor in β-epithelialsodium channel (β-ENaC) and γ- ENaC subunits. In contrast,the ENaC α-subunit abundance was significantly increasedin Gal 3–/– as compared with WT mice (100 ± 24 in WT vs.232 ± 17 arbitrary units in Gal 3–/–; n = 5 animals in eachgroup; p < 0.001) (Figure 4). In the inner medulla, there was

Table III. Assessment of renal function in WT and Gal 3–/– mice: clearance study in mice perfused with 150 mM NaHCO3

Gal 3–/–, galectin 3 null mutant mice; GFR, glomerular filtration rate; MAP, mean arterial pressure; N, number of mice; NS, not significant; WT, wild type.The data represent mean ± SEM.

MAP (mmHg)Arterial [HCO3

–] (mM)Arterial PCO2(mmHg) Arterial pH GFR (μL/min)

Urinary flowrate (μL/min)

HCO3– excretion

rate (nmol/min)

WT (N = 4) 120 ± 6 32 ± 2 31 ± 4 7.62 ± 0.03 289 ± 41 1.5 ± 0.1 38 ± 8

Gal 3–/– (N = 4) 91 ± 2 30 ± 2 31 ± 5 7.50 ± 0.07 312 ± 64 1.9 ± 0.2 38 ± 6

p < 0.003 NS NS NS NS NS NS

Table IV. Number of glomeruli and kidney weights in WT and Gal 3–/– mice

Gal 3–/–, galectin 3 null mutant mice; N, number of mice; NS, not significant; WT, wild type.The data represent mean ± SEM.

Number of glomeruli per kidney Kidney weight (mg)

WT 12,565 ± 325 164 ± 6 (N = 13)

Gal 3–/– 11,184 ± 489 191 ± 7 (N = 16)

p < 0.04 p < 0.006

Fig. 3. Systolic blood pressure in conscious mice. Tail-cuff blood pressure was measured during 6 months in mice submitted to low (0.038% Na) or high (3% Na) sodium diet. Control WT and age-matched Gal 3–/– mice were either maintained on low sodium diet (WT, N = 5; Gal 3–/–, N = 8) or on high sodium diet (WT, N = 4; Gal 3–/–, N = 9). Bars represent mean ± SEM.

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no significant difference in the abundance of sodium-potassium-chloride cotransporter 1 (NKCC1), the basolateralNa+–K+–2Cl– cotransporter of the inner medullary CD.

Discussion

The aim of this study was to gain insight into the role ofgalectin 3 in kidney functions. For this purpose, adult Gal 3null mutant mice were analyzed. It is remarkable that Gal 3–/–mice develop normally, look healthy, and do not exhibitgross phenotypic abnormalities. Under basal conditions,they have normal blood pressure levels and plasma ion con-centrations. No alteration in acid–base equilibrium (thatcould attest for dysfunctions of intercalated cells of the CD)was seen in Gal 3–/– mice. It should be noted that we founddetectable expression of galectin 3 in both principal andintercalated cells of the CD of adult WT mice. However, thedistribution of galectin 3 in these cells was variable in differ-ent regions of the kidney. Although B-intercalated cellsalways contained Gal 3, A-intercalated cells in the cortexwere largely negative, but A-cells in the inner stripe and innermedulla were clearly positive. This implies not only a subtypespecific, but also a regional difference in Gal 3 expression inintercalated cells. Previous studies have also describedregional differences in the patterns of lectin binding of inter-calated cells that may reflect functional differences in variouskidney regions (LeHir et al., 1982; Brown et al., 1985;Holthofer et al., 1988). However, after acute bicarbonate load-ing, WT and Gal 3–/– mice had the same degree of metabolicalkalosis and identical bicarbonate urinary excretion rates.Thus, the present results did not provide evidence for alteredfunctions of intercalated cells of the CD despite an absenceof Gal 3. These results do not exclude the possibility thatadaptation of CD intercalated cells to chronic changes in theacid–base status might be altered in Gal 3–/– mice, but fur-ther more extensive work will be needed to test this hypothesis.

Two sets of abnormalities were discovered in Gal 3–/–mice. First, 15- to 31-week-old Gal 3–/– mice exhibited alower number of glomeruli (~11%). This observation isopposite to what might have been expected from the experi-ments on ex vivo addition of galectin 3 to developing ure-teric buds (Winyard et al., 1997), thus underlying thedifficulty in extrapolating such data to in vivo situations.However, the formal possibility remains that the defect we

report only appeared after birth. Interestingly, the smallreduction in the number of nephrons in Gal 3–/– mice wasaccompanied by an increase (~17%) in kidney weight, with-out glomerular histological damage. The GFR was equiva-lent in mutant and WT mice. Taken together, normal GFRand the reduced number of glomeruli imply an increasedsingle nephron GFR, that is, a hyperfiltration state. It hasbeen suggested that such a condition results in an increasein the glomerular pressure that may lead to glomerularinjury and may favor evolution towards renal failure inhumans as well as in animals (Hostetter, 2003). However,this does not seem to be the case in Gal 3–/– mice becausethe histological appearance of the kidney was normal. Fur-thermore, 1-year-old Gal 3–/– mice had normal plasmaurea and creatinine levels. In other words, there was no signof chronic renal insufficiency. A small reduction of thenumber of nephrons has also been proposed to favor therise in blood pressure levels, in particular in face of anincreased salt intake (Johnson et al., 2002). Our results inGal 3–/– mice do not support such hypothesis becausechronic high-salt diet did not increase blood pressure inthese animals.

Second, the use of metabolic challenges (i.e., acutesodium chloride infusion or chronic salt loading or restric-tion) allowed us to unravel a subtle renal chloride wastingsyndrome in Gal 3–/– mice. Indeed, clearance experimentsin mice perfused with an isotonic sodium chloride solutionrevealed an increased renal ability to excrete chloride (asNaCl plus KCl) in Gal 3–/– mice. Furthermore, low-saltdiet (for 5 months) allowed us to unmask signs of ECF vol-ume contraction, as suggested by the bioimpedancemethod, which was presumably the consequence of mildchronic NaCl urinary losses. Consistent with a reducedECF volume, Gal 3–/– mice kept under either low- or normal-salt diet, exhibited much higher plasma aldosterone levelsthan WT mice, which may be interpreted as a compensa-tory mechanism in response to sodium chloride loss. Highaldosterone concentrations are likely to be the cause ofhigher α-ENaC expression observed in the kidney of Gal 3–/–mice since this subunit is known to be specifically up-regu-lated by aldosterone in the kidney (Escoubet et al., 1997).Results of blood pressure levels following high-sodium dietfor 5 months may also be interpreted in light of the renalsodium chloride loss of Gal 3–/– mice. In the high salt dietconditions, Gal 3–/– mice clearly exhibited lower blood

Fig. 4. Immunoblots of homogenates of renal cortex of WT and Gal 3–/– mice. The abundance of ENaC α-subunit was enhanced in Gal 3–/– mice (p < 0.001). There was no difference in the abundance of α-Na-K/ATPase, ENaC β- and γ-subunits, and β-actin. Each lane of these 8.5% polyacrylamide gels was loaded with 35 μg of protein prepared from five separate animals.

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pressure values than WT mice. This indicates that Gal 3–/–mice do not notably increase their ECF volume and conse-quently their blood pressure in response to a high-sodiumdiet stimulus. In this way, Gal 3–/– mice appeared resistantto salt.

Excessive chloride excretion may originate from a defectin ion transporters responsible for its reabsorption or anoveractivity of transporters involved in NaCl and KCl secre-tion. Alternatively, it could be because of defective chloridefluxes through cellular junctions. Because galectin 3 wasfound in the TAL of Henle’s loop and along the CD, severalhypotheses may account for the chloride loss in Gal 3–/–mice. In the TAL, the apical Na/K/2Cl cotransporter(BSC1/NKCC2) controls apical chloride uptake from thelumen of the tubule. Chloride is then extruded at the baso-lateral pole of the TAL cells through chloride channel(ClC)-KB channels and KCl cotransport, whereas potas-sium is recycled back to the tubular fluid by the apicalpotassium channel ROMK. Mutations in NKCC2, inROMK, and in ClC-KB (and in its regulatory protein Barttin)have been identified in patients with Bartter’s syndrome(Hebert, 2003). Patients with Bartter’s syndrome exhibit uri-nary sodium chloride wasting with hypokalemia and meta-bolic alkalosis (Hebert, 2003). Gal 3–/– mice have somefeatures of mild Bartter syndrome but without alterations inbicarbonate or potassium plasma concentrations. It may beworth considering Gal 3 as a possible candidate gene in casesof mild salt loosing syndromes where no mutations of theclassical candidate genes have been found. In the CD, para-cellular chloride reabsorption occurs principally throughtight junctions, whereas its secretion depends on the basolat-eral Na/K/Cl cotransporter (NKCC1) and on apical CICs.The abundance of NKCC1 protein was comparable in WTand in Gal 3–/– mice, but this observation does not rule outits possible enhanced transport activity in Gal 3–/– mice.Mutations in the with no lysine kinase 4 (WNK4) gene havebeen recently shown to be the cause of pseudohypoaldoster-onism type II, an inherited form of hypertension (Wilsonet al., 2001). Interestingly the mutation of WNK4 increasesparacellular chloride permeability, thus designating WNK4as a novel regulatory pathway for the paracellular chlorideshunt (Yamauchi et al., 2004). It might be proposed thatGal 3, at the level of tight junctions, may contribute to thispathway. In the absence of Gal 3, paracellular chloride reab-sorption might be reduced, which should lead to a pheno-type close to that observed in Gal 3–/– mice.

In conclusion, Gal 3–/– mice exhibit renal abnormalities,including an hyperfiltration state and a chloride wastingsyndrome that led to abnormal ECF balance in response tochanges in salt diet. They may be related to direct (but yetunknown) consequences of Gal 3 knockout. The mild phe-notype observed here may also be because of compensa-tions by other galectins, an aspect that has not beenexplored in this study.

Materials and methods

Animals

The Gal 3 null mutation was generated using the WW6 EScell line which was originally derived from a blastocyst

from a mouse line carrying an inert genetic marker (Ioffeet al., 1995). This ES cell line is exceptionally stable andeasy to handle; however, it has a mixed genetic composition(75% 129/Sv, 20%C57BL/6J, 5% SJL). The Gal 3–/– mouseline was established by standard procedure using 129/Svanimals to breed the original chimerae (Colnot et al.,1998a). All mutant animals used in this study were obtainedafter two additional backcrosses on the 129/Sv background.The corresponding mixed WT counterparts were used ascontrols. The genetic background of all individualsincluded in these experiments is 98% 129/Sv, 1.6% C57B6/6J, and 0.4% SJL. Mice were fed a standard diet (0.3% Na)or were challenged by chronic diets containing either low(0.038% Na) or high (3% Na) sodium. These diets (UAR,Epinay sur Orge, France) were given starting from the timeof weaning (at 3 weeks) until 5 months later. Only maleswere included in this study. Handling and studies have beenperformed in accordance with INSERM rules for animalexperiments.

Kidney histology and immunofluorescence studies

Histology of the kidneys was assessed by routine examina-tion of kidney sections after hematoxylin eosin and periodicacid schiff staining. Galectin 3 was immunolocalized in sectionsof kidneys of WT mice, as previously described (Pu et al.,2001). Briefly, after in vivo aortic perfusion of 2%paraformaldehyde in phosphate buffer, frozen kidney sec-tions were incubated with a polyclonal anti-galectin 3 anti-body (Hubert et al., 1995) diluted 1:400 overnight at 4°C.To identify tubular segments (Pu et al., 2001), antibodiesagainst AQP 2 (specific for PCs in renal CDs) or againsturomucoid (URO, specific for the TAL) were used follow-ing the incubation with anti-galectin 3. Secondary antibod-ies were as previously described (goat anti-rabbit GAR-Cy3or the Fab fragment, GAR-Alexa, or donkey anti-goatFITC) (Pu et al., 2001). Furthermore, cryosections (5 μm)of control and galectin 3 knockout mouse kidney werethaw-mounted onto slides and rehydrated in phosphate-buffered saline (PBS). Sections were blocked for 10 min in1% bovine serum albumin (BSA)/PBS and double-stainedfor 2 h at room temperature using rabbit-anti-galectin 3antibody, diluted 1:400 and affinity purified chicken-anti-PP antibody (B1-subunit), diluted 1:20. The secondary anti-bodies used were goat anti-rabbit Alexa red (1:800) anddonkey anti-chicken FITC, diluted 1:200, respectively. Theywere applied for 45 min. After washing in PBS, sectionswere mounted in Prolong anti-fade reagent (MolecularProbes, Eugene, OR).

Immunoelectron microscopy

Small pieces of WT mouse kidney cortex and medulla (fixedby perfusion with 2% paraformaldehyde) were dehydratedthrough a graded series of ethanol, infiltrated with LRWhite, medium hard grade (EMS, Ft. Washington, PA),and embedded in LR White in gelatin capsules overnight at50°C. Ultrathin sections were cut on a Reichert Ultracut Eultramicrotome and collected on Formvar coated nickelgrids. Sections were blocked on drops of 5% normal goatserum/1% BSA/PBS for 10 min at room temperature andincubated on drops of primary antibody or diluent alone.

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The antibody used was rabbit-anti-galectin 3 (1:400). Afterrinsing, the sections were incubated for a further 45 min withgoat anti-rabbit immunoglobulin G (IgG) coupled to 15 nmgold particles (commercial solution diluted 1:20) and rabbitanti-chicken IgG coupled to 10 nm gold (1:20 dilution)(Ted Pella Inc., Redding, CA). Sections were poststainedfor 5 min on drops of 2% aqueous uranyl acetate in methyl-cellulose, rinsed in distilled water, and examined in a PhilipsCM 10 transmission electron microscope.

Renal function study

Mice were weighed and anesthetized with thiobutabarbital(inactin, 100 mg/kg, i.p.) and ketamine (100 mg/kg, i.m.)and their rectal temperature was maintained at 37°C on aservo-controlled heated surgical table. Tracheostomy wasperformed and a PE-50 catheter hand-drawn to the appro-priate size was inserted into the right femoral artery forblood pressure measurements (Gould transducer, Eastlake,OH) and blood sampling. The right external jugular veinwas cannulated with a hand-drawn PE-50 catheter for con-tinuous infusion of isotonic saline (0.9 mL/h/100 g bodyweight) or 150 mM NaHCO3 (1.8 mL/h/100 g body weight)containing [methoxy-3H]inulin (Perkin Elmer, Boston,MA; priming dose of 12.5 μCi followed by 30 μCi/h). Aftera 45–60 min equilibration period, a timed 45–60 min urinesample was collected through a bladder catheter under par-affin oil equilibrated with water or with a 100 mM Hepes-25 mM NaHCO3 solution and bubbled with 5% CO2 toavoid any CO2 loss. Blood samples were taken from thefemoral artery in heparinized glass capillaries before (~15 μL)and after (200 μL) the clearance period. At the end of theexperiments, the kidneys were excised, decapsulated, towelblotted, and weighed. Some kidneys were placed in 5 N HClat 37°C for 90 min, then in distilled water overnight at 4°C,and the number of glomeruli was counted under microscopeobservation as described previously (Bonvalet et al., 1977).The urine volume was determined gravimetrically. The con-centrations of ions in plasma and urine samples were mea-sured by flame photometry (Elex 6361, Eppendorf; sodiumand potassium) and with the Monarch apparatus (Instru-mentation Laboratory, Lexington, MA; chloride). Plasmabicarbonate concentration was calculated from measure-ments of arterial pH and PCO2 (ABL520 Radiometer,Copenhagen, Denmark). When mice were perfused with150 mM NaHCO3 to induce acute metabolic alkalosis, theurinary bicarbonate concentration was measured immedi-ately after the end of the clearance period with a flow-through microfluorometer (NanoFlo, World PrecisionInstruments, Sarasota, FL; Zheliaskov et al., 2000). Theradioactivity of [methoxy-3H]inulin in plasma and urine sam-ples (1–2 μL) was measured by liquid scintillation counting(LKB, 1209 rackbeta) for determination of the GFR.

Body fluid volumes

Body water compartments were estimated by multiple fre-quency bioelectrical impedance (MFBIA) as previouslydescribed for rats (Yokoi et al., 2001). Data were collectedusing the Analycor XF (Spengler, Cachan, France) ana-lyzer. Measurements were made using the standard tetrapo-lar technique with current injection on the right fore limb

and the left hind limb and sensing electrodes on the left forelimb and the right hind limb. Platinum subdermal elec-trodes (Grass Instrument Company, Quincy, MA) wereused as injection and sensing electrodes. Mice were anesthe-tized by halothane and placed on a nonconductive surfaceto eliminate interference of electrical induction. The bodyweight was recorded to the nearest 0.1 g. Body length wasmeasured from the narium to the pelvic-caudal junction.Data were analyzed with software IMP BO4 (Spengler).Twenty frequencies were measured ranging from 1 to 300 kHz.As demonstrated in humans (De Lorenzo et al., 1997) andrats (Yokoi et al., 2001) impedance spectral data were fit tothe Cole model, and ECW and ICW volumes were inferredby using model electrical resistance terms RE and RI,respectively, in an equation derived from Hanai mixturetheory. Extracellular and intracellular resistivity needed toresolve Hanaï equations were based on human databecause the corresponding data for mice were unavailable,which precludes determination of absolute values of bodywater volumes but provides arbitrary units that are reliablefor comparing different conditions.

Systolic blood pressure measurements in conscious mice

Arterial systolic blood pressure was measured by the tail-cuff plethysmography method in trained conscious miceplaced in a warming restrainer. Tail-cuff pressure, detectedby a pressure transducer (SP844, SensoNor asa, Horten,Norway), and tail arterial pulsations, detected by a piezo-electric pulse sensor (RTBP096, Kent Scientific Corpora-tion, Torrington, CT), were amplified by a signal amplifierQazap 92204-02 (Bionic Instruments, Phymep S.A., Paris,France). Signals were processed and displayed by means ofa PowerLab/4SP program (ADInstruments, Phymep SA,Paris, France). Arterial pressure was defined as the tail-cuffinflation pressure at which the waveform was extinguished.For all mice, measurements were repeated for 3 days, between10 a.m. and 1 p.m. Each day, approximately 10 consecu-tive inflation cycles were performed and final blood pres-sure was calculated by averaging successful readings.Blood pressure was first measured 3 weeks after startingeach specific diet and then every month for 5 months.

Plasma aldosterone concentration

Plasma aldosterone concentration was measured by radio-immunoassay (ALDOCTK-2, DiaSorin s.r.l., Vercelli,Italy).

Electrophoresis and immunoblotting of membrane proteins

The cortex and inner medulla of mice were homogenized ina medium composed of 125 mM sucrose, 12 mM TrizmapH 7.4, 0.1 mM 4-(2-aminoethyl)-benzenesulfonyl fluoridehydrochlorine (AEBSF), and 5 μg/mL leupeptin, andstored at −80°C until use. The homogenates were solubi-lized at room temperature for 20 min in Laemmli mediumcontaining (final concentrations) 62.5 mM Tris–HCl pH6.8, 5% SDS, 100 mM DTT, and 10% glycerol. Sodiumdodecyl sulphate-polyacrylamide gel electrophoresis (SDS–PAGE) was performed with solubilized homogenates (35 μgprotein) and prestained molecular weight markers (Sigma,LaVerpilliere, France) on 8.5% polyacrylamide minigels

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(Mini Protean II, Bio-Rad, Marnes la Coquette, France).Proteins were subsequently transferred from the gels to nitro-cellulose membranes by electrophoresis (Mini Trans BlotModule, Bio-Rad). Loading and transfer efficiency were sys-tematically monitored by Ponceau red staining of the nitro-cellulose membranes. The membranes were incubated withrabbit polyclonal antibodies raised against NKCC1 (Chemicon,Temecula, CA), Na/K-ATPase α-subunit (Djelidi et al., 1997),and α–, β–, and γ-subunits of the ENaC (Djelidi et al., 1997)and with a β-actin mouse monoclonal antibody (Sigma-Aldrich Fine Chemicals, LaVerpilliere, France) and then tothe secondary antibodies [peroxidase linked anti-rabbit Igand anti-mouse Ig (Bio-Rad)]. Quantification of each bandwas performed using NIH Image software. Quantification ofβ-actin was used as a control to compare loading and transferefficiency. Results of band densities are expressed relative tothat of β-actin.

Statistics

Results are expressed as means ± SEM. Statistical signifi-cance between experimental groups was assessed by Stu-dent’s t test or by one- or multi-way analysis of variance(ANOVA) completed by a t test, as appropriate.

Acknowledgments

The in vivo assessment of renal function and blood pressurelevels have been made in the facilities (the CEFI, for Centred’Explorations Fonctionnelles Intégrées dedicated tomouse phenotyping) developed by the Institut Federatif deRecherches IFR 02 Claude Bernard of the Xavier BichatMedical School. This work was supported by INSERMand partly by the Association pour la Recherche sur le Can-cer (number 4680) and Ligue contre le cancer, comité deParis, allocated to Françoise Poirier, and by NIH grantnumber DK42956 (Dennis Brown). We thank MaryMcKee for excellent technical assistance with electronmicroscopy and immunofluorescence microscopy. We alsothank R.C. Hughes for continuous support and scientificdiscussions throughout the course of this work.

Abbreviations

CCD, cortical collecting duct; CD, collecting duct; ECF,extracellular fluid; ECW, extracellular water; ENaC, epithelialsodium channel; Gal 3–/–, galectin 3 null mutant mice;GFR, glomerular filtration rate; ICW, intracellular water;NKCC, sodium-potassium-chloride cotransporter; PBS,phosphate-buffered saline; PC, principal cell; PP, protonpump; TAL, thick ascending limb of Henle’s loop; TDL,thin descending limb of Henle’s loop; WNK4, with nolysine kinase 4; WT, wild type.

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