NDT Advance Access originally published online on June 9, 2006
Nephrology Dialysis Transplantation 2006 21(9):2391-2398; doi:10.1093/ndt/gfl255
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© The Author [2006]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org
Original Articles: Experimental Nephrology
The tight junction proteins claudin-7 and -8 display a different subcellular localization at Henle's loops and collecting ducts of rabbit kidney
Department of Physiology, Biophysics and Neurosciences, Center for Research and Advanced Studies (Cinvestav), Mexico DF, Mexico
Correspondence and offprint requests to: L. Gonzalez-Mariscal, Department of Physiology, Biophysics and Neurosciences, Center for Research and Advanced Studies (Cinvestav), Ave. Instituto Politecnico Nacional 2508, Mexico, DF 07360, Mexico. Email: lorenza{at}fisio.cinvestav.mx
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Abstract |
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Background. The tight junction (TJ) regulates the passage of ions and molecules through the paracellular pathway. In multicellular organisms, epithelial sheets function as a barrier between a variety of environments and the internal media. Therefore, TJs are required to control the passage of diverse molecules in different epithelia. The mammalian nephron constitutes a particularly relevant model of this diversity, since the paracellular transport in this organ is significantly different along the various tubular segments. Here, we have analysed the distribution of claudins-7 and -8 in Henle's loops and collecting ducts isolated from rabbit kidneys.
Methods. Renal segments were manually isolated from newborn and adult rabbit kidneys and processed for immunofluorescence. The distribution of claudins-7 and -8 was studied by confocal microscopy.
Results. The localization of claudins-7 and -8 along Henle's loops and collecting ducts is remarkably different. While claudin-8 displays a clear cell border distribution in Henle's segment, claudin-7 shows a non-specific cytosolic staining. Moreover, in the collecting ducts, claudin-8 localizes at the TJ region, while claudin-7 shows a basolateral staining. This pattern is present from the newborn stage. The distribution of claudins along the mammalian kidney has been found to vary in different mammalian species. Accordingly, in the rabbit, we have found the expression of claudin-8 at the descending and ascending thin limbs of Henle, a distribution that differs from that found in the mouse by others.
Conclusion. In the rabbit Henle's loop, claudin-8 is present at the cellular borders of the descending and ascending thin limbs, while claudin-7 displays no specific labelling. Instead, at the collecting duct, both claudins are present but exhibit a different subcellular distribution.
Keywords: claudins; Henle's loop; kidney; paracellular permeability; tight junctions
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Introduction |
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In multicellular organisms different body compartments are separated by epithelial sheets. In them, cells are characterized for being polarized and for having an intercellular junctional complex. Polarity is a crucial feature of epithelia since it allows the vectorial transport of substances between the external environment or the lumen of internal compartments and the internal milieu of the individual. The junctional complex is constituted by structures that associate the lateral membrane of neighbouring cells. In vertebrates, at the upper portion of the lateral membrane, the intercellular space is sealed by the tight junction (TJ). This structure that encircles epithelial and endothelial cells, regulates the passage of ions and molecules through the paracellular pathway and maintains cell polarity by blocking the free diffusion of lipids and proteins within the plasma membrane between the apical and basolateral surfaces [1].
TJs seal the paracellular pathway through interactions established at the intercellular space by the extracellular loops and segments of the integral TJ proteins occludin, claudins and JAM. Claudins constitute a family of different members found across a wide range of multicellular animals. Claudins are proteins of 20–25 kDa that traverse the membrane four times and expose two loops towards the intercellular space. The first loop ranges from 45–55 residues and contains a WGLWCC signature motif, while the second loop has a length of 10–21 residues. The cytoplasmic tail of claudins has 21–44 residues and ends in a PDZ-binding motif, capable of association with PDZ domains present in TJ scaffolding proteins like MUPP1, ZO-1, ZO-2 and ZO-3. The transfection of claudins into cells lacking TJs, such as fibroblasts, triggers the appearance of filaments that resemble those of TJs in epithelial cells [2], and freeze-fracture replicas of cells labelled by immunogold for claudins show gold particles associated with TJ strands, thus indicating that claudins are the building blocks of TJ filaments.
TJs behave as charged and size-selective channels that lack directional rectification [3]. The evidence that claudins influence size selectivity comes from the observation that, in claudin-5 null mice, the blood brain barrier becomes leaky to a small-size marker (562 Da) while it still restricts the passage of a 1862 Da marker [4]. The role of claudins in the formation of TJ channels was proposed after observing that claudin-16, whose expression is restricted to the thick ascending loop (TAL) of Henle, a region where 60% of the filtered magnesium is reabsorbed [5], is mutated in a familial magnesium-wasting disease [6]. Further information on the role of claudins has been gained by introducing certain claudins in different cell culture models. These experiments have indicated that while claudins like 4, 8 and 11 discriminate against cations [7,8], others, exemplified by claudins-2 and -15, display an opposite charge selectivity [7,9]. These differential effects are due to the charges of the residues found on the extracellular loops of claudins.
The expression of a particular set of claudins is crucial for the permeation of solutes across epithelia, via the paracellular pathway. In consequence, tissues exposed to different environments express diverse groups of claudins. The nephron constitutes a particularly interesting model for the study of claudin expression, since each nephron segment differs in histological structure and function. For example, the proximal tubule, where an important reabsorption of filtered components takes place, expresses claudin-2 [10,11]. In contrast, the distal nephron that maintains high transtubular cationic gradients expresses claudins-7 and -8 [11–13].
The presence of claudins at Henle's segments has been difficult to analyse in frozen tissue sections because of the extreme narrowness of this nephron segment. Hence, conflicting results have been reported. With regard to claudin-7, studies done in the mouse have identified it exclusively at the descending thin limb (DTL) [12], while in pork and rat it was only observed at the thick ascending limb (TAL) [13]. However, the precise distribution of claudin-7 at the reported Henle's segments is difficult to assess because in the frozen sections either a slight or diffuse expression of claudin-7 is detected. In the case of claudin-8, the situation becomes even more complex because in employing the same animal species, mouse, some authors have found this claudin at the DTL [12] whereas others have observed it only at the ascending thin limb (ATL) [11].
Here, we have tried to solve this discrepancy by analysing the expression of claudins-7 and -8 in manually dissected Henle's segments because this preparation allows a more accurate identification of each segment and renders a better view of the distribution pattern of TJ proteins [10,14]. Furthermore, claudins-7 and -8 have been reported to be expressed in the exact same nephron segments but localized at different subcellular sites [12]. However, the latter observation has not been clearly documented. Therefore, here we have analysed, in isolated collecting segments, the subcellular distribution of claudins-7 and -8.
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Animals
New Zealand male white rabbits (2.0–2.5 kg) were maintained on diet science and water ad libitum, in our animal facilities, at 22–24°C with 50–55% humidity. Newborn rabbits (5 days old) were kept with the dam until the experimentation date. Care and handling of the animals were done in accordance with internationally accepted procedures.
Isolation of rabbit renal tubules
Rabbit tubules were manually dissected as previously described [14].
Immunofluorescence
The isolated tubules were fixed and processed for immunofluorescence as previously described [14]. The following primary antibodies were used: rabbit polyclonals against claudin-7 (Zymed, 349100; 1 µg/ml, South San Francisco, CA, USA) and claudin-8 (antibody 933, dilution 1:100; Invitrogen Custom Antibody Service, Carlsbad, CA, USA, generously provided by Dr Alan Yu of the University of Southern California) or goat polyclonals against aquaporin 1 (AQP1) (Santa Cruz Biotechnology sc-9879; dilution 1:100) and Rab-11 (Santa Cruz Biotechnology sc-6565; dilution 1:50), or mouse monoclonal antibodies against occludin (Zymed 33-1500, 2 µg/ml), cytokeratin (Ck) 8 (Boehringer 1238-817; 5 µg/ml; Mannheim, Germany), glycoprotein gp135 (a generous gift of Dr George Ojakian, NY State University), Rab-5 (BD Biosciences 610281, dilution 1:50) and LAMP-1 (Santa Cruz Biotechnology sc-17768; dilution 1:50). As secondary antibodies, we employed an FITC-conjugated goat anti-rabbit IgG (Zymed 65-6511; dilution 1:100), a TRITC-conjugated antimouse IgG developed in goat (Sigma T 5393; dilution 1:100) and a donkey anti-goat polyclonal conjugated to TRITC (Jackson Immunoresearch Laboratories 705-025-003; dilution 1:100). Fluorescence was examined in a confocal microscope (Leica SP2) with a krypton–argon laser. For the colocalization of claudin-7 with Rab-5, Rab-7 and LAMP-1, single optical sections were analysed.
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Claudin-7 displays a cytoplasmic distribution along the different segments of Henle's loop
In this study, we have employed AQP1, CK 8 and gp135 as specific markers of the following segments of Henle's loop: DTL [11], ATL and TAL, respectively [15]. We started the study by observing the background immunofluorescence of the isolated tubules treated only with the secondary antibodies. Figure 1 illustrates the weak immunofluorescence detected with the FITC-conjugated (A) anti-rabbit antibody and (B) the anti-mouse or (C) anti-goat TRITC-conjugated polyclonals. Figure 2 shows how, at the Henle's segment that is positive for AQP1 and therefore was identified as DTL, claudin-7 gives a rather diffuse cytoplasmic staining sometimes concentrated in punctae and not a junctional associated pattern. A similar situation is encountered at the ATL segment, identified for its positive CK 8 label. In the lower panel of Figure 2, two portions of a single isolated Henle's segment are observed. The upper thin one is negative for gp135 and positive for claudin-7, while the lower portion, which is remarkably wider, can be identified as TAL for its positive gp135 staining. This portion displays a conspicuously weaker cytosolic staining for claudin-7.
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In order to determine whether the punctate staining of claudin-7 corresponds to a vesicular compartment, we made double-staining experiments with claudin-7 and one of the following antibodies: the small GTPases Rab-5 and Rab-11 and LAMP-1, which respectively label early endosomes, recycling endosomes and late endosomes or lysosomes [16]. Figures 3, 4 and 5 illustrate how no claudin-7 is found to localize in the tested endosomal and lysosomal compartments. We next tested whether the low cytoplasmic staining of claudin-7 is similar to that detected in other renal segments, known to be negative to claudin-7. Figure 6 illustrates the claudin-7 signal obtained in a proximal isolated rabbit tubule, which resembles that of the thin Henle's limb treated only with a secondary FITC-conjugated antibody (Figure 1) or those receiving claudin-7 and the secondary fluorescent antibody (Figure 2). Taken together, these results suggest no specific labelling of claudin-7 at Henle's loop.
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Claudin-8 has a junctional distribution at the descending and ascending limbs of Henle's loop
The images in Figure 7 reveal the clear junctional presence of claudin-8 at the DTL segment, identified for its positive AQP1 signal and at the ATL tubule recognized for being positive to CK 8 staining. In contrast, the lower panel of the figure shows a wider tubule that corresponds to the TAL segment identified for its positive gp135 staining that is nonetheless negative to claudin-8 (asterisk). This particular tubule that continues to a thinner section, as observed in the image (arrowhead), is positive to claudin-8 staining at the cell borders and negative for gp135, hence indicating its identification as an ATL segment.
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Claudins-7 and -8 are conspicuously present at the collecting ducts; however, while claudin-8 has a junctional distribution, claudin-7 shows a basolateral pattern
It has been suggested that claudins-7 and -8 are present in the same renal tubular segments. Therefore, we next analysed the distribution of these claudins in the collecting ducts of rabbits. We co-stained the renal tubules with claudins and occludin, since the latter gives a precise localization of TJs. The upper panel of Figure 8 illustrates how claudin-7 and occludin do not colocalize, as the former displays a basolateral distribution while occludin is restricted to the TJ region.
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The lower panel of Figure 8 shows how, in contrast to claudin-7, claudin-8 colocalizes with occludin at the TJ. Taken together, these results indicate that, at the tighter segment of the rabbit nephron, both, claudins-7 and -8 are expressed albeit at different locations of the plasma membrane. The tubules shown in Figure 8 were dissected from newborn animals. A similar distribution of claudins-7 and -8 is observed in adult tissue, indicating that this particular distribution of claudins-7 and -8 is established early in the rabbit nephron.
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To our knowledge, only two studies have explored in detail the subcellular distribution of claudin-7 along the mammalian kidney. In the mouse study performed by Li et al. [12], the presence of claudin-7 at the DTL section is assessed by its colocalization with AQP1, while in the porcine kidney cortex, the study by Alexandre et al. [13] locates claudin-7 at the TAL, identified by its positive BSC1 staining (Na/K/2Cl cotransporter). However, the disadvantage of both of these studies lies in the fact that the distribution of claudins was analysed in frozen sections. In the latter, it is particularly difficult to disclose the junctional proteins in Henle's segments due to their thinness. Here, we show that, in the rabbit, claudin-7 displays a cytoplasmic pattern similar to that detected when only the secondary fluorescent antibody is employed and to that obtained in the proximal tubule, a segment that does not express claudin-7 [12]. Furthermore, the punctuate staining obtained with claudin-7 did not colocalize with that of the cellular vesicular compartments.
The distribution of claudin-8 in different Henle's segments, has only being analysed to our knowledge, in the mouse. However, conflicting results have been obtained. Hence, while the work of Kiuchi-Saishin et al. [11] did not detect claudin-8 at the DTL identified with AQP1, the work of Li et al. [12] reported claudin-8 staining at the late segments of the DTL. In our manually dissected rabbit tubules, we have been able to observe long Henle's segments positive for AQP1 staining that display claudin-8 at the cellular borders; therefore we conclude that at least in the rabbit, claudin-8 is present in the DTL segments. With regard to the ATL, Kiuchi-Saishin et al. [11] have found that renal cells positive for the chloride channel-K1, express a junctional pattern of claudin-8. Instead, the work of Li et al. [12] did not report claudin-8 at the cells that express this label. In the rabbit, claudin-8 depicts a characteristic chicken-fence pattern in the Henle's tubules, identified as ATL segments by CK 8. In agreement with the results obtained in the mouse we did not detect claudin-8 at the TAL identified by the gp135 staining.
In a recent study performed in LLC-PK1 cells, the overexpression of claudin-7 generated a decrease in the paracellular conductance to Cl–, concurrent with an increased paracellular conductance to Na+; therefore this claudin is regarded as a paracellular channel to Na+ and a barrier to Cl–. The ATL of Henle's loop is known to be leaky to sodium and to present the highest chloride permeability of any nephron segment [17]. Therefore, it is not surprising to observe that at the ATL segment, claudin-7 does not localize at the junctional region and instead gives a diffuse cytoplasmic staining that resembles that found in the DTL, a region known to be impermeable to Na+.
Claudin-7 was previously found expressed in the distal and collecting tubules [12,13]. This expression seems a paradox, since the distal portion of the nephron generates transtubular gradients of 1:30 (lumen:peritubular) for Na+ [18], which would be dissipated if a passive back-leak through the paracellular pathway is present. The basolateral localization of claudin-7 that we observed in the collecting ducts, was previously mentioned but not shown in the nephron [12]. Furthermore, in the airway epithelium [19] and in the rat uterus [20], claudin-7 has been found at the basolateral membrane. Therefore, the presence of claudin-7 at the basolateral surface of the collecting tubules probably indicates that, in this region, claudin-7 does not act as a paracellular cation channel that could obstruct the development of the steep transtubular cation gradients, instead claudin-7 could have another function. Speculatively, claudin-7 could be participating in cell–cell and cell–matrix adhesion at the basolateral membrane.
Claudin-8 in MDCK cells acts primarily as a cation barrier [21], and therefore its expression at the TJ in the collecting duct might aid in the maintenance of the steep transtubular cation gradients found in the distal nephron. In contrast, its expression in the ATL, as shown by us in the rabbit and by Kiuchi-Saishin et al. [11] in the mouse is more difficult to explain since this segment is leaky to sodium. However, the ATL segments are also highly permeable to Cl–, and claudin-8 has been found not to reduce the paracellular permeability of anions [21].
This work, hence indicates that with regard to claudins, it is of utmost importance not only to detect the expression of the protein but also to determine the region of the cell where the protein concentrates. In the nephron, this characteristic can significantly be better achieved in isolated tubules than in frozen sections. We think that until claudins are better understood, the physiological consequences of their presence at sites different from TJs remain speculative.
Conflict of interest statement. None declared.
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This work was supported by grants G34511M and 45691-Q from the National Research Council of Mexico (CONACyT).
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References |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
- Anderson JM, Cereijido M. Evolution of ideas on the tight junction. In: Cereijido M, Anderson JM. Tight Junctions, CRC Press, Boca Raton: 2001; 1–18
- Furuse M, Sasaki H, Fujimoto K, Tsukita S. A single gene product, claudin-1 or -2, reconstitutes tight junction strands and recruits occludin in fibroblasts. J Cell Biol 1998; 143: 391–401
[Abstract/Free Full Text] - Cereijido M, Robbins ES, Dolan WJ, Rotunno CA, Sabatini DD. Polarized monolayers formed by epithelial cells on a permeable and translucent support. J Cell Biol 1978; 77: 853–880
[Abstract/Free Full Text] - Nitta T, Hata M, Gotoh S et al. Size-selective loosening of the blood-brain barrier in claudin-5-deficient mice. J Cell Biol 2003; 161: 653–660
[Abstract/Free Full Text] - Quamme GA, de Rouffignac C. Epithelial magnesium transport and regulation by the kidney. Front Biosci 2000; 5: D694–D711[Web of Science][Medline]
- Simon DB, Lu Y, Choate KA et al. Paracellin-1, a renal tight junction protein required for paracellular Mg2+ resorption. Science 1999; 285: 103–106
[Abstract/Free Full Text] - Van Itallie CM, Fanning AS, Anderson JM. Reversal of charge selectivity in cation or anion-selective epithelial lines by expression of different claudins. Am J Physiol Renal Physiol 2003; 285: F1078–F1084
[Abstract/Free Full Text] - Van Itallie C, Rahner C, Anderson JM. Regulated expression of claudin-4 decreases paracellular conductance through a selective decrease in sodium permeability. J Clin Invest 2001; 107: 1319–1327[Web of Science][Medline]
- Amasheh S, Meiri N, Gitter AH et al. Claudin-2 expression induces cation-selective channels in tight junctions of epithelial cells. J Cell Sci 2002; 115: 4969–4976
- Reyes JL, Lamas M, Martin D et al. The renal segmental distribution of claudins changes with development. Kidney Int 2002; 62: 476–487[CrossRef][Web of Science][Medline]
- Kiuchi-Saishin Y, Gotoh S, Furuse M et al. Differential expression patterns of claudins, tight junction membrane proteins, in mouse nephron segments. J Am Soc Nephrol 2002; 13: 875–886
[Abstract/Free Full Text] - Li WY, Huey CL, Yu AS. Expression of claudin-7 and -8 along the mouse nephron. Am J Physiol Renal Physiol 2004; 286: F1063–F1071
[Abstract/Free Full Text] - Alexandre MD, Lu Q, Chen YH. Overexpression of claudin-7 decreases the paracellular Cl– conductance and increases the paracellular Na+ conductance in LLC-PK1 cells. J Cell Sci 2005; 118: 2683–2693
[Abstract/Free Full Text] - Gonzalez-Mariscal L, Namorado MC, Martin D et al. Tight junction proteins ZO-1, ZO-2, and occludin along isolated renal tubules. Kidney Int 2000; 57: 2386–2402[CrossRef][Web of Science][Medline]
- Piepenhagen PA, Peters LL, Lux SE, Nelson WJ. Differential expression of Na(+)-K(+)-ATPase, ankyrin, fodrin, and E-cadherin along the kidney nephron. Am J Physiol 1995; 269: C1417–C1432
- Ivanov AI, Nusrat A, Parkos CA. Endocytosis of epithelial apical junctional proteins by a clathrin-mediated pathway into a unique storage compartment. Mol Biol Cell 2004; 15: 176–188
[Abstract/Free Full Text] - Imai M. Function of the thin ascending limb of Henle of rats and hamsters perfused in vitro. Am J Physiol 1977; 232: F201–F209[Medline]
- Liddle GW. Aldosterone antagonists. AMA Arch Intern Med 1958; 102: 998–1004[Web of Science][Medline]
- Coyne CB, Gambling TM, Boucher RC, Carson JL, Johnson LG. Role of claudin interactions in airway tight junctional permeability. Am J Physiol Lung Cell Mol Physiol 2003; 285: L1166–L1178
[Abstract/Free Full Text] - Mendoza-Rodriguez CA, Gonzalez-Mariscal L, Cerbon M. Changes in the distribution of ZO-1, occludin, and claudins in the rat uterine epithelium during the estrous cycle. Cell Tissue Res 2005; 319: 315–330[CrossRef][Web of Science][Medline]
- Yu AS, Enck AH, Lencer WI, Schneeberger EE. Claudin-8 expression in Madin-Darby canine kidney cells augments the paracellular barrier to cation permeation. J Biol Chem 2003; 278: 17350–17359
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Accepted in revised form: 10. 4.06
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