Glycobiology Advance Access originally published online on September 15, 2005
Glycobiology 2006 16(1):36-45; doi:10.1093/glycob/cwj035
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Exploring the role of galectin 3 in kidney function: a genetic approach
2 INSERM U426, 16 rue Henri Huchard, 75870 Paris Cedex 18, France; 3 IFR 2 Claude Bernard, 16 rue Henri Huchard, 75870 Paris Cedex 18, France; 4 Université Paris 7, 2 place Jussieu, 75005 Paris, France; 5 Program in Membrane Biology/Renal Unit, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114; 6 Service de Néphrologie, Hôpital Xavier Bichat, 46 rue Henri Huchard, 75870 Paris Cedex 18, France; 7 Centre d’Explorations Fonctionnelles Intégrées, IFR 2 Claude Bernard, 16 rue Henri Huchard, 75870 Paris Cedex 18, France; 8 Association Claude Bernard, Centre de recherche de génétique et pathologie moléculaire de l’hématopoièse, 16 rue Henri Huchard, 75870 Paris Cedex 18, France; 9 Institut Jacques Monod, CNRS UMR 7592, Universités Paris 6 and Paris 7, 2 place Jussieu, 75251 Paris Cedex 05, France; 10 INSERM U478, 16 rue Henri Huchard, 75870 Paris Cedex 18, France; and 11 Equipe EA 3102, Service d’Anatomopathologie, Hôpital Robert Debré, 75019 Paris, France
1 To whom correspondence should be addressed; e-mail: poirier{at}ijm.jussieu.fr
Received on May 30, 2005; revised on August 31, 2005; accepted on September 1, 2005
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Galectin 3 belongs to a family of glycoconjugate-binding proteins that participate in cellular homeostasis by modulating cell growth, adhesion, and signaling. We studied adult galectin 3 null mutant (Gal 3–/–) and wild-type (WT) mice to gain insights into the role of galectin 3 in the kidney. By immunofluorescence, 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. Compared to WT mice, Gal 3–/– mice had ~11% fewer glomeruli (p < 0.04), associated with kidney hypertrophy (p < 0.006). In clearance experiments, urinary chloride excretion was found to be higher in Gal 3–/– than in WT mice (p < 0.04), but there was no difference in urinary bicarbonate excretion, in glomerular filtration, or urinary flow rates. Under chronic low sodium diet, Gal 3–/– mice had lower extracellular fluid (ECF) volume than WT mice (p < 0.05). Plasma aldosterone concentration was higher in Gal 3–/– than in WT mice (p < 0.04), which probably caused the observed increase in
-epithelial sodium channel (-ENaC) protein abundance in the mutant mice (p < 0.001). Chronic high sodium diet resulted paradoxically in lower blood pressure (p < 0.01) in Gal 3–/– than in WT. We conclude that Gal 3–/– mice have mild renal chloride loss, which causes chronic ECF volume contraction and reduced blood pressure levels. Key words: blood pressure / body fluid volumes / hyperfiltration / Bartter’s like syndrome / null mutant mouse
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Introduction |
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Lectins are proteins specifically binding to glycoconjugates. Lectins play important roles in several normal and pathological processes through largely unknown molecular mechanisms. Among lectins, galectins are specific for beta-galactoside derivatives and share a unique structure that is entirely different from other types of lectins (Barondes et al., 1994a
,b). The mammalian galectin gene family includes 12 members (Houzelstein et al., 2004). Galectins are relatively small proteins (14–30 kDa), and, despite the lack of signal peptide, they can be found both inside and outside the cells depending on the cell type and on the stage of differentiation (Barondes et al., 1994a,b). This latter unusual property may at least partly account for the broad spectrum of potential functions that have been ascribed to galectins. Hence, there is a large body of evidence showing that galectins could act not only as modulators of cell–cell or cell–substratum interactions but also as apoptotic (or antiapoptotic) molecules (Hughes, 2001; Liu et al., 2002). Galectin 3 is normally expressed in the developing human (Winyard et al., 1997) and mouse (Bullock et al., 2001) kidney where it has been proposed to play a role in fetal collecting duct (CD) morphogenesis since exogenous addition of galectin 3 to embryonic mouse kidney explants was shown to inhibit branching of the ureteric bud (Bullock et al., 2001). More recently, it has been proposed that galectin 3 participates in the functional adaptation to metabolic acidosis of intercalated cells of the renal CD (Hikita et al., 2000). The A- and B-intercalated cells in the collecting tubules of the kidney are responsible for the final acidification or alkalinization of the urine. Metabolic acidosis is proposed to remodel B-intercalated (bicarbonate-secreting) cells into A-intercalated (acid-secreting) cells (Al-Awqati, 2003). A similar remodeling of cell polarity has been characterized in detail in an immortalized cell line, and this process requires the deposition of hensin in the extracellular matrix. Hensin is a 230 kDa extracellular glycoprotein that can induce, in its polymerized form, terminal differentiation of renal intercalated cells cultured in vitro (Hikita et al., 2000). The signal for terminal differentiation of intercalated cells appears to be the secretion of galectin 3 (Hikita et al., 2000). Once outside the cells, galectin 3 binds to and promotes polymerization of hensin, and the hensin-galectin 3 complex is able to induce the conversion of B-like intercalated cells into A-like intercalated cells in vitro (Hikita et al., 2000). Little is known about the localization and the role of galectin 3 in the kidney in vivo. In humans, galectin 3 expression decreases after birth and was noted to be restricted to a subset of cells in the CD in infants (Winyard et al., 1997). Renal expression of galectin 3 mRNA and protein was shown to be upregulated in acute renal failure in the rat, thus suggesting a role in acute tubular injury and the subsequent regeneration stage (Nishiyama et al., 2000). In the kidney of normal rat, a very weak galectin 3 signal was found to be confined to distal tubules (Nishiyama et al., 2000). However, the precise cell type was not identified with established markers in these experiments.
To assess the role of galectin 3 in vivo, we have studied Gal 3 null mutant mice (Gal 3–/–) (Colnot et al., 1998a). These Gal 3–/– mice are viable and fertile but several defects associated with this mutation have been observed and 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 the absence of galectin 3 leads to an accelerated glomerulopathy after streptozotocin-induction of diabetes, which was attributed to disruption of the capacity of galectin 3 to bind toxic advanced glycation end products (Pugliese et al., 2001). The aim of this study was to search for a function of galectin 3 in the kidney glomerular and tubular functions in adult mice. We found that Gal 3–/– mice have a reduced number of nephrons without renal insufficiency, and that they show an increased renal ability to excrete chloride leading to extracellular fluid (ECF) volume contraction and lower blood pressure after challenge with high salt diet as compared to wild-type (WT) mice.
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Kidney histology and immunolocalization of Galectin 3
By standard histological examination, the kidneys of Gal 3–/– mice seemed normal and did not appear to be different from those of WT mice (data not shown). Cell-specific expression of galectin 3 on kidney sections is illustrated in Figure 1. In the cortex of WT mice, a minority of tubules appeared to express galectin 3 and, as expected, no signal was present over sections of Gal 3–/– mice (data not shown). In the cortical CD (Figure 1A and C), a majority of cells had positive galectin 3 signal, suggesting predominant expression in principal cells (PCs) that represent ~60% of the cells in this tubular segment in the mouse (Kim et al., 1999
). This is confirmed by colocalization of galectin 3 and aquaporin 2 (AQP 2), a marker of PCs (Figure 1A, panel b). Staining with anti-proton pump (PP) antibodies revealed that most B-type intercalated cells in the cortex also contained galectin 3, but the staining intensity was lower than in PCs (Figure 1C, cortex). In contrast, many A-type intercalated cells in the cortex were not stained, or only weakly-stained for galectin 3, although an occasional A-cell was more brightly stained. A similar pattern was seen in the outer stripe of the outer medulla, where PCs were positive and most A-intercalated cells were unstained (Figure 1C, outer stripe). This pattern of staining in the cortical and outer medullary CDs was confirmed by immuno-electron microscopy (Figure 2) which revealed unstained A-intercalated cells adjacent to heavily-stained PCs in cortical and outer medullary (outer stripe) CDs. Cortical B-intercalated cells were moderately stained (Figure 2). In contrast, both A-intercalated cells and PCs in the inner stripe and in the inner medulla were equally well-stained for galectin 3 (Figure 1C, inner stripe). Galectin-3 positive cells were also present in tubules that express uromucoid (Figure 1A, panel c), indicating that galectin 3 is also expressed in the thick ascending limb of Henle’s loop (TAL). In contrast, proximal tubules and glomeruli were consistently negative. Regions of transition between the S3 segment of the proximal tubule and the thin descending limb of Henle’s loop (TDL) of Henle showed that TDLs were also positive for galectin 3 (Figure 1B). These results indicate that galectin 3 in adult mouse kidney is expressed in CD PCs and B-intercalated cells, as well as A-intercalated cells in the inner stripe and the inner medulla. Thick ascending limbs of Henle are also positive. Galectin 3 seemed mainly localized inside the cells (both cytoplasm and nucleus in some cases) without apparent extracellular staining.
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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 values of protein, sodium, potassium, chloride, and bicarbonate between WT and Gal 3–/– mice (Table I). Clearance experiments were performed under two different conditions. First, mice were perfused with isotonic saline. As summarized in Table II, the glomerular filtration and urinary flow rates and the sodium and potassium excretion rates were comparable in the two strains of mice. However, Gal 3–/– mice had a significantly higher (p < 0.04) chloride excretion rate than WT mice (Tables I–III). Note that there was no significant difference 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 minus chloride urinary concentration was the same in both groups of mice (64 ± 15 in WT vs. 51 ± 16 mM in Gal 3–/–, n = 6 in each group, NS), it appears that Gal 3–/– mice excreted more NaCl plus KCl than WT mice. Second, mice were perfused with 150 mM NaHCO3 to measure urinary bicarbonate excretion rates with accuracy. WT and Gal 3–/– mice had the same degree of metabolic alkalosis at the end of the clearance experiments (Table III). There was no difference in 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 significantly lower mean arterial pressure than WT in this experimental series (p < 0.003; Table III).
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The number of glomeruli per kidney was ~11% lower in Gal 3–/– than in WT mice (p < 0.04; Table IV), but the kidney weight was higher in Gal 3–/– than in WT mice (p < 0.006; Table IV). These results indicate that there was a lower number of nephrons with compensatory kidney hypertrophy in Gal 3–/– mice. However, this did not lead to chronic renal insufficiency in aging Gal 3–/– mice, as estimated by measurements of plasma creatinine (38 ± 1 vs. 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 phosphate (1.9 ± 0.3 vs. 2. 6 ± 0.2 mM) and glucose (8 ± 1 vs. 10 ± 2 mM) concentrations were also comparable in WT and Gal 3–/– old animals.
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Systolic blood pressure in conscious mice
In these series, WT and Gal 3–/– mice were maintained under low (0.038% Na) or high (3% Na) sodium intake for a period of 5 months after weaning and noninvasive tail-cuff blood pressure was measured each month in conscious animals 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 values obtained in each group during the study were pooled. As shown in Figure 3, there was no significant difference in the blood pressure values between Gal 3–/– and WT mice submitted to the low sodium diet. In contrast, chronic high sodium diet allowed to reveal a striking and unexpected difference, because systolic blood pressure levels were then significantly lower in Gal 3–/– (141 ± 3 mmHg, N = 9 animals) than in WT (156 ± 5 mmHg, N = 4 animals; p < 0.01) mice (Figure 3).
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Body fluid volumes
Body weight and body fluid volumes were assessed in 29-week-old WT and Gal 3–/– mice maintained on low or high sodium 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 arbitrary units vs. 9.8 ± 0.6 in WT; p < 0.04) than age-matched WT mice. Intracellular water (ICW) content did not differ between Gal 3–/– and WT mice. In the high sodium diet conditions, there was a high mortality rate at 7 months of age in the WT group so that only two WT mice could be studied (body weight: 28.2 and 34.0 g). In contrast, Gal 3–/– mice did not die and eight mice could be examined (body weight, 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 mice with normal diet or after 6 months on a low or high sodium diet. Plasma aldosterone concentrations were significantly higher in Gal 3–/– than in WT mice maintained on both the low (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 in WT; p < 0.04) sodium diet. For mice maintained on a high sodium diet, only two values of plasma aldosterone concentration (64 and 52 pg/mL) could be obtained in WT mice because of the high mortality rate as mentioned above. By contrast, nine values were obtained in Gal 3–/– mice (97 ± 22 pg/mL). Altogether elevated plasma aldosterone levels in Gal 3–/– mice are consistent with the renal salt wasting and ECF volume contraction described above in these animals.
Quantification of transport proteins
Since Gal 3–/– mice excreted more NaCl plus KCl than WT mice, the abundance of some transport proteins expressed in the CDs was assessed by western blot experiments in mice subjected to the standard diet. In the renal cortex, Figure 4 shows that there was no difference in the abundance of the Na/K-ATPase -subunit nor in ß-epithelial sodium channel (ß-ENaC) and 
- ENaC subunits. In contrast, the ENaC -subunit abundance was significantly increased in Gal 3–/– as compared with WT mice (100 ± 24 in WT vs. 232 ± 17 arbitrary units in Gal 3–/–; n = 5 animals in each group; p < 0.001) (Figure 4). In the inner medulla, there was no significant difference in the abundance of sodium-potassium-chloride cotransporter 1 (NKCC1), the basolateral Na+–K+–2Cl– cotransporter of the inner medullary CD.
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Discussion |
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The aim of this study was to gain insight into the role of galectin 3 in kidney functions. For this purpose, adult Gal 3 null mutant mice were analyzed. It is remarkable that Gal 3–/– mice develop normally, look healthy, and do not exhibit gross phenotypic abnormalities. Under basal conditions, they have normal blood pressure levels and plasma ion concentrations. No alteration in acid–base equilibrium (that could attest for dysfunctions of intercalated cells of the CD) was seen in Gal 3–/– mice. It should be noted that we found detectable expression of galectin 3 in both principal and intercalated cells of the CD of adult WT mice. However, the distribution of galectin 3 in these cells was variable in different regions of the kidney. Although B-intercalated cells always contained Gal 3, A-intercalated cells in the cortex were largely negative, but A-cells in the inner stripe and inner medulla were clearly positive. This implies not only a subtype specific, but also a regional difference in Gal 3 expression in intercalated cells. Previous studies have also described regional differences in the patterns of lectin binding of intercalated cells that may reflect functional differences in various kidney regions (LeHir et al., 1982
; Brown et al., 1985; Holthofer et al., 1988). However, after acute bicarbonate loading, WT and Gal 3–/– mice had the same degree of metabolic alkalosis and identical bicarbonate urinary excretion rates. Thus, the present results did not provide evidence for altered functions of intercalated cells of the CD despite an absence of Gal 3. These results do not exclude the possibility that adaptation of CD intercalated cells to chronic changes in the acid–base status might be altered in Gal 3–/– mice, but further 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 a lower number of glomeruli (~11%). This observation is opposite to what might have been expected from the experiments on ex vivo addition of galectin 3 to developing ureteric buds (Winyard et al., 1997), thus underlying the difficulty in extrapolating such data to in vivo situations. However, the formal possibility remains that the defect we report only appeared after birth. Interestingly, the small reduction in the number of nephrons in Gal 3–/– mice was accompanied by an increase (~17%) in kidney weight, without glomerular histological damage. The GFR was equivalent in mutant and WT mice. Taken together, normal GFR and the reduced number of glomeruli imply an increased single nephron GFR, that is, a hyperfiltration state. It has been suggested that such a condition results in an increase in the glomerular pressure that may lead to glomerular injury and may favor evolution towards renal failure in humans as well as in animals (Hostetter, 2003). However, this does not seem to be the case in Gal 3–/– mice because the histological appearance of the kidney was normal. Furthermore, 1-year-old Gal 3–/– mice had normal plasma urea and creatinine levels. In other words, there was no sign of chronic renal insufficiency. A small reduction of the number of nephrons has also been proposed to favor the rise in blood pressure levels, in particular in face of an increased salt intake (Johnson et al., 2002). Our results in Gal 3–/– mice do not support such hypothesis because chronic high-salt diet did not increase blood pressure in these animals.
Second, the use of metabolic challenges (i.e., acute sodium chloride infusion or chronic salt loading or restriction) allowed us to unravel a subtle renal chloride wasting syndrome in Gal 3–/– mice. Indeed, clearance experiments in mice perfused with an isotonic sodium chloride solution revealed an increased renal ability to excrete chloride (as NaCl plus KCl) in Gal 3–/– mice. Furthermore, low-salt diet (for 5 months) allowed us to unmask signs of ECF volume contraction, as suggested by the bioimpedance method, which was presumably the consequence of mild chronic NaCl urinary losses. Consistent with a reduced ECF volume, Gal 3–/– mice kept under either low- or normal-salt diet, exhibited much higher plasma aldosterone levels than WT mice, which may be interpreted as a compensatory mechanism in response to sodium chloride loss. High aldosterone concentrations are likely to be the cause of higher -ENaC expression observed in the kidney of Gal 3–/– mice since this subunit is known to be specifically up-regulated by aldosterone in the kidney (Escoubet et al., 1997). Results of blood pressure levels following high-sodium diet for 5 months may also be interpreted in light of the renal sodium chloride loss of Gal 3–/– mice. In the high salt diet conditions, Gal 3–/– mice clearly exhibited lower blood pressure values than WT mice. This indicates that Gal 3–/– mice do not notably increase their ECF volume and consequently their blood pressure in response to a high-sodium diet stimulus. In this way, Gal 3–/– mice appeared resistant to salt.
Excessive chloride excretion may originate from a defect in ion transporters responsible for its reabsorption or an overactivity of transporters involved in NaCl and KCl secretion. Alternatively, it could be because of defective chloride fluxes through cellular junctions. Because galectin 3 was found in the TAL of Henle’s loop and along the CD, several hypotheses 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 the lumen of the tubule. Chloride is then extruded at the basolateral pole of the TAL cells through chloride channel (ClC)-KB channels and KCl cotransport, whereas potassium is recycled back to the tubular fluid by the apical potassium channel ROMK. Mutations in NKCC2, in ROMK, 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 urinary sodium chloride wasting with hypokalemia and metabolic alkalosis (Hebert, 2003). Gal 3–/– mice have some features of mild Bartter syndrome but without alterations in bicarbonate or potassium plasma concentrations. It may be worth considering Gal 3 as a possible candidate gene in cases of mild salt loosing syndromes where no mutations of the classical candidate genes have been found. In the CD, paracellular chloride reabsorption occurs principally through tight junctions, whereas its secretion depends on the basolateral Na/K/Cl cotransporter (NKCC1) and on apical CICs. The abundance of NKCC1 protein was comparable in WT and in Gal 3–/– mice, but this observation does not rule out its possible enhanced transport activity in Gal 3–/– mice. Mutations in the with no lysine kinase 4 (WNK4) gene have been recently shown to be the cause of pseudohypoaldosteronism type II, an inherited form of hypertension (Wilson et al., 2001). Interestingly the mutation of WNK4 increases paracellular chloride permeability, thus designating WNK4 as a novel regulatory pathway for the paracellular chloride shunt (Yamauchi et al., 2004). It might be proposed that Gal 3, at the level of tight junctions, may contribute to this pathway. In the absence of Gal 3, paracellular chloride reabsorption might be reduced, which should lead to a phenotype close to that observed in Gal 3–/– mice.
In conclusion, Gal 3–/– mice exhibit renal abnormalities, including an hyperfiltration state and a chloride wasting syndrome that led to abnormal ECF balance in response to changes in salt diet. They may be related to direct (but yet unknown) consequences of Gal 3 knockout. The mild phenotype observed here may also be because of compensations by other galectins, an aspect that has not been explored in this study.
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Materials and methods |
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Animals
The Gal 3 null mutation was generated using the WW6 ES cell line which was originally derived from a blastocyst from a mouse line carrying an inert genetic marker (Ioffe et al., 1995
). This ES cell line is exceptionally stable and easy to handle; however, it has a mixed genetic composition (75% 129/Sv, 20%C57BL/6J, 5% SJL). The Gal 3–/– mouse line was established by standard procedure using 129/Sv animals to breed the original chimerae (Colnot et al., 1998a). All mutant animals used in this study were obtained after two additional backcrosses on the 129/Sv background. The corresponding mixed WT counterparts were used as controls. The genetic background of all individuals included 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 time of weaning (at 3 weeks) until 5 months later. Only males were included in this study. Handling and studies have been performed in accordance with INSERM rules for animal experiments.
Kidney histology and immunofluorescence studies
Histology of the kidneys was assessed by routine examination of kidney sections after hematoxylin eosin and periodic acid schiff staining. Galectin 3 was immunolocalized in sections of 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 sections were incubated with a polyclonal anti-galectin 3 antibody (Hubert et al., 1995) diluted 1:400 overnight at 4°C. To identify tubular segments (Pu et al., 2001), antibodies against AQP 2 (specific for PCs in renal CDs) or against uromucoid (URO, specific for the TAL) were used following the incubation with anti-galectin 3. Secondary antibodies were as previously described (goat anti-rabbit GAR-Cy3 or the Fab fragment, GAR-Alexa, or donkey anti-goat FITC) (Pu et al., 2001). Furthermore, cryosections (5 µm) of control and galectin 3 knockout mouse kidney were thaw-mounted onto slides and rehydrated in phosphate-buffered saline (PBS). Sections were blocked for 10 min in 1% bovine serum albumin (BSA)/PBS and double-stained for 2 h at room temperature using rabbit-anti-galectin 3 antibody, diluted 1:400 and affinity purified chicken-anti-PP antibody (B1-subunit), diluted 1:20. The secondary antibodies used were goat anti-rabbit Alexa red (1:800) and donkey anti-chicken FITC, diluted 1:200, respectively. They were applied for 45 min. After washing in PBS, sections were mounted in Prolong anti-fade reagent (Molecular Probes, Eugene, OR).
Immunoelectron microscopy
Small pieces of WT mouse kidney cortex and medulla (fixed by perfusion with 2% paraformaldehyde) were dehydrated through a graded series of ethanol, infiltrated with LR White, medium hard grade (EMS, Ft. Washington, PA), and embedded in LR White in gelatin capsules overnight at 50°C. Ultrathin sections were cut on a Reichert Ultracut E ultramicrotome and collected on Formvar coated nickel grids. Sections were blocked on drops of 5% normal goat serum/1% BSA/PBS for 10 min at room temperature and incubated on drops of primary antibody or diluent alone. The antibody used was rabbit-anti-galectin 3 (1:400). After rinsing, the sections were incubated for a further 45 min with goat anti-rabbit immunoglobulin G (IgG) coupled to 15 nm gold particles (commercial solution diluted 1:20) and rabbit anti-chicken IgG coupled to 10 nm gold (1:20 dilution) (Ted Pella Inc., Redding, CA). Sections were poststained for 5 min on drops of 2% aqueous uranyl acetate in methylcellulose, rinsed in distilled water, and examined in a Philips CM 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 a servo-controlled heated surgical table. Tracheostomy was performed and a PE-50 catheter hand-drawn to the appropriate size was inserted into the right femoral artery for blood pressure measurements (Gould transducer, Eastlake, OH) and blood sampling. The right external jugular vein was cannulated with a hand-drawn PE-50 catheter for continuous infusion of isotonic saline (0.9 mL/h/100 g body weight) 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). After a 45–60 min equilibration period, a timed 45–60 min urine sample was collected through a bladder catheter under paraffin oil equilibrated with water or with a 100 mM Hepes-25 mM NaHCO3 solution and bubbled with 5% CO2 to avoid any CO2 loss. Blood samples were taken from the femoral artery in heparinized glass capillaries before (~15 µL) and after (200 µL) the clearance period. At the end of the experiments, the kidneys were excised, decapsulated, towel blotted, and weighed. Some kidneys were placed in 5 N HCl at 37°C for 90 min, then in distilled water overnight at 4°C, and the number of glomeruli was counted under microscope observation as described previously (Bonvalet et al., 1977). The urine volume was determined gravimetrically. The concentrations of ions in plasma and urine samples were measured by flame photometry (Elex 6361, Eppendorf; sodium and potassium) and with the Monarch apparatus (Instrumentation Laboratory, Lexington, MA; chloride). Plasma bicarbonate concentration was calculated from measurements of arterial pH and PCO2 (ABL520 Radiometer, Copenhagen, Denmark). When mice were perfused with 150 mM NaHCO3 to induce acute metabolic alkalosis, the urinary bicarbonate concentration was measured immediately after the end of the clearance period with a flow-through microfluorometer (NanoFlo, World Precision Instruments, Sarasota, FL; Zheliaskov et al., 2000). The radioactivity of [methoxy-3H]inulin in plasma and urine samples (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 frequency bioelectrical impedance (MFBIA) as previously described for rats (Yokoi et al., 2001). Data were collected using the Analycor XF (Spengler, Cachan, France) analyzer. Measurements were made using the standard tetrapolar technique with current injection on the right fore limb and the left hind limb and sensing electrodes on the left fore limb and the right hind limb. Platinum subdermal electrodes (Grass Instrument Company, Quincy, MA) were used as injection and sensing electrodes. Mice were anesthetized by halothane and placed on a nonconductive surface to eliminate interference of electrical induction. The body weight was recorded to the nearest 0.1 g. Body length was measured 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) and rats (Yokoi et al., 2001) impedance spectral data were fit to the Cole model, and ECW and ICW volumes were inferred by using model electrical resistance terms RE and RI, respectively, in an equation derived from Hanai mixture theory. Extracellular and intracellular resistivity needed to resolve Hanaï equations were based on human data because the corresponding data for mice were unavailable, which precludes determination of absolute values of body water volumes but provides arbitrary units that are reliable for 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 mice placed in a warming restrainer. Tail-cuff pressure, detected by a pressure transducer (SP844, SensoNor asa, Horten, Norway), and tail arterial pulsations, detected by a piezoelectric pulse sensor (RTBP096, Kent Scientific Corporation, Torrington, CT), were amplified by a signal amplifier Qazap 92204-02 (Bionic Instruments, Phymep S.A., Paris, France). Signals were processed and displayed by means of a PowerLab/4SP program (ADInstruments, Phymep SA, Paris, France). Arterial pressure was defined as the tail-cuff inflation pressure at which the waveform was extinguished. For all mice, measurements were repeated for 3 days, between 10 a.m. and 1 p.m. Each day, approximately 10 consecutive inflation cycles were performed and final blood pressure was calculated by averaging successful readings. Blood pressure was first measured 3 weeks after starting each specific diet and then every month for 5 months.
Plasma aldosterone concentration
Plasma aldosterone concentration was measured by radioimmunoassay (ALDOCTK-2, DiaSorin s.r.l., Vercelli, Italy).
Electrophoresis and immunoblotting of membrane proteins
The cortex and inner medulla of mice were homogenized in a medium composed of 125 mM sucrose, 12 mM Trizma pH 7.4, 0.1 mM 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochlorine (AEBSF), and 5 µg/mL leupeptin, and stored at –80°C until use. The homogenates were solubilized at room temperature for 20 min in Laemmli medium containing (final concentrations) 62.5 mM Tris–HCl pH 6.8, 5% SDS, 100 mM DTT, and 10% glycerol. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS–PAGE) was performed with solubilized homogenates (35 µg protein) and prestained molecular weight markers (Sigma, LaVerpilliere, France) on 8.5% polyacrylamide minigels (Mini Protean II, Bio-Rad, Marnes la Coquette, France). Proteins were subsequently transferred from the gels to nitrocellulose membranes by electrophoresis (Mini Trans Blot Module, Bio-Rad). Loading and transfer efficiency were systematically monitored by Ponceau red staining of the nitrocellulose membranes. The membranes were incubated with rabbit polyclonal antibodies raised against NKCC1 (Chemicon, Temecula, CA), Na/K-ATPase -subunit (Djelidi et al., 1997), and –, ß–, and -subunits of the ENaC (Djelidi et al., 1997) and with a ß-actin mouse monoclonal antibody (Sigma-Aldrich Fine Chemicals, LaVerpilliere, France) and then to the secondary antibodies [peroxidase linked anti-rabbit Ig and anti-mouse Ig (Bio-Rad)]. Quantification of each band was performed using NIH Image software. Quantification of ß-actin was used as a control to compare loading and transfer efficiency. Results of band densities are expressed relative to that of ß-actin.
Statistics
Results are expressed as means ± SEM. Statistical significance between experimental groups was assessed by Student’s t test or by one- or multi-way analysis of variance (ANOVA) completed by a t test, as appropriate.
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TopAbstractIntroductionResultsDiscussionMaterials and methodsAcknowledgmentsReferences |
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The in vivo assessment of renal function and blood pressure levels have been made in the facilities (the CEFI, for Centre d’Explorations Fonctionnelles Intégrées dedicated to mouse phenotyping) developed by the Institut Federatif de Recherches IFR 02 Claude Bernard of the Xavier Bichat Medical School. This work was supported by INSERM and partly by the Association pour la Recherche sur le Cancer (number 4680) and Ligue contre le cancer, comité de Paris, allocated to Françoise Poirier, and by NIH grant number DK42956 (Dennis Brown). We thank Mary McKee for excellent technical assistance with electron microscopy and immunofluorescence microscopy. We also thank R.C. Hughes for continuous support and scientific discussions throughout the course of this work.
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Abbreviations |
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CCD, cortical collecting duct; CD, collecting duct; ECF, extracellular fluid; ECW, extracellular water; ENaC, epithelial sodium 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, proton pump; TAL, thick ascending limb of Henle’s loop; TDL, thin descending limb of Henle’s loop; WNK4, with no lysine kinase 4; WT, wild type
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