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Am J Physiol Heart Circ Physiol 287: H203-H208, 2004. First published February 19, 2004; doi:10.1152/ajpheart.01237.2003
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Glycosaminoglycan polymerization may enable osmotically inactive Na+ storage in the skin

Jens Titze,1 Mehdi Shakibaei,2 Markus Schafflhuber,1 Gundula Schulze-Tanzil,2 Markus Porst,1 Karl H. Schwind,3 Peter Dietsch,4 and Karl F. Hilgers1

1Department of Medicine IV, Friedrich-Alexander-University Erlangen-Nürnberg, D-90471 Nürnberg; Departments of 2Anatomy and 4Biochemistry, Charité-Universitätsmedizin Berlin, Campus Benjamin Franklin, D-14195 Berlin; and 3Department of Chemistry and Physics, Federal Center for Meat Research, D-95326 Kulmbach, Germany

Submitted 29 December 2003 ; accepted in final form 13 February 2004


   ABSTRACT
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Osmotically inactive skin Na+ storage is characterized by Na+ accumulation without water accumulation in the skin. Negatively charged glycosaminoglycans (GAGs) may be important in skin Na+ storage. We investigated changes in skin GAG content and key enzymes of GAG chain polymerization during osmotically inactive skin Na+ storage. Female Sprague-Dawley rats were fed a 0.1% or 8% NaCl diet for 8 wk. Skin GAG content was measured by Western blot analysis. mRNA content of key dermatan sulfate polymerization enzymes was measured by real-time PCR. The Na+ concentration in skin was determined by dry ashing. Skin Na+ concentration during osmotically inactive Na+ storage was 180–190 mmol/l. Increasing skin Na+ coincided with increasing GAG content in cartilage and skin. Dietary NaCl loading coincided with increased chondroitin synthase mRNA content in the skin, whereas xylosyl transferase, biglycan, and decorin content were unchanged. We conclude that osmotically inactive skin Na+ storage is an active process characterized by an increased GAG content in the reservoir tissue. Inhibition or disinhibition of GAG chain polymerization may regulate osmotically inactive Na+ storage.

hypertension; extracellular matrix; chondroitin synthase; elongation enzymes

SODIUM IS PRESENT NOT ONLY in the extracellular fluid (ECF) volume, where it exerts osmotic effects, but also at other sites in the body, where it is osmotically inactive. The phenomenon of osmotically inactive Na+ storage has been described in humans (6, 15) consuming a high-NaCl diet during long-term experiments and has been confirmed experimentally in animals (14). The site at which Na+ is deposited is not certain, although bone has been suggested. We recently demonstrated that osmotically inactive Na+ storage in rats takes place largely in the skin. However, which cutaneous structure might serve as an osmotically inactive Na+ reservoir is unclear.

Mixed connective tissues have been reported to contain large amounts of Na+ (2). Glycosaminoglycans (GAGs) consist of alternating copolymers of uronic acids and amino sugars. GAGs are negatively charged polyanions. Their negative charge density increases with their sulfatation grade (18). Most animal cells are surrounded by an interstitial fluid of relatively constant cation composition, dominated by Na+, which is present at ~140 mmol/l ECF. However, chondrocytes are surrounded by an ECF with an ionic composition that is altered by the negative charge density of GAGs in the extracellular matrix (5, 9, 10). The negatively charged GAGs attract cations and repel anions, resulting in extracellular cartilage Na+ concentration of 250–350 mmol/l (7) and extracellular osmolality of 350–450 mmol/l (17). Linear sulfated GAGs are classified as the galactosaminoglycans chondroitin sulfate (CS) and dermatan sulfate (DS) or the glucosaminoglycans heparan sulfate (HS) and heparin (12). Both types of GAG chains are attached to their respective core proteins, such as biglycan or decorin, through a GAG-protein linkage region (GlcA-Gal-Gal-Xyl-O-Ser). Xylosyl transferase, which transfers xylose to the GAG core protein, is thus the key enzyme of GAG chain initiation. HS, CS, and DS chains are attached to their common GAG-protein linkage region. The enzyme HS polymerase is responsible for the repeating disaccharide regions glucuronic acid (GlcA) and N-acetylglucosamine. In contrast, CS and DS chains are elongated through alternate addition of GlcA and N-acetylgalactosamine. N-acetylgalactosamine is attached to GlcA through the enzyme 1,3-acetylgalactosaminyltransferase, whereas 4-N-acetylgalactosaminyltransferase is responsible for the GalNAc 1,4 attachment to GlcA. The GAG chain polymerization enzyme chondroitin synthase has 1,3- and 1,4-linkage activity.

We hypothesized that osmotically inactive skin Na+ storage might be an active process that, similar to cartilage, includes the modification of GAG content in skin. Increased GAG chain initiation and/or elongation might increase the negative charge density in the connective tissue. This could lead to osmotically inactive Na+ storage. Therefore, we investigated the mRNA content of GAG initiation and elongation enzymes in the skin of rats fed a low- or high-NaCl diet.


   MATERIALS AND METHODS
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 MATERIALS AND METHODS
RESULTS
 DISCUSSION
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Animal experiments. All animal experiments were done according to American Society of Physiology guidelines and were approved by local government authorities. Twenty 9- to 10-wk-old female Sprague-Dawley rats (Charles River) were divided into two groups with similar body weights, 193.3 ± 3.6 g (group 1) and 198.0 ± 4.0 (group 2), and fed regular rat chow (0.61% NaCl) for 1 wk. From week 2, the rats were fed different diets: group 1 was fed a virtually Na+-free diet (<0.1% NaCl) for 8 consecutive weeks, and group 2 was fed a high-Na+ diet (8% NaCl) for 8 wk. Both Na+ diets contained 0.95% Ca2+ and 0.70% K+. After the animals were fed their specified diets for 8 wk, they were anesthetized with methohexital (100 mg/kg body wt ip), the left femoral arteries were catheterized, and blood samples were taken before the rats were killed. Small slices of skin and femur cartilage (~30–50 mg/sample) were shock frozen for further biochemical analysis. Six of 10 carcasses per group were skinned completely to determine total skin Na+ and water content in the animals. In the remaining four carcasses in each group, we removed a piece of skin and subcutaneous tissue from the back to investigate Na+ and water content relative to skin weight.

Skin ashing and electrolytes. Skin samples were desiccated at 90°C for 72 h. Skin water content was calculated from the difference between wet weight and dry weight. After the samples were dry ashed at 190°C for 24 h and 600°C for further 48 h, the skin ashes were dissolved in 20 ml of 10% HNO3. Na+ concentrations in blood samples were measured with a flame photometer (model EFIX, Eppendorf, Hamburg, Germany). Na+ concentrations in the dissolved ashes were measured with flame photometry (model 3100, Perkin Elmer, Rodgau, Germany).

Western blotting. Cells and matrix proteins from shock-frozen skin and cartilage samples were extracted with lysis buffer (50 mM Tris·HCl, pH 7.2, 150 mM NaCl, 1% Triton X-100, 1 mM sodium orthovanadate, 50 µg/ml sodium pyrophosphate, 100 mM sodium fluoride, 0.01% aprotinin, 4 µg/ml pepstatin A, 10 µg/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride) on ice for 30 min. For immunoblotting, equal amounts of total proteins were separated on 5% or 7.5% SDS-polyacrylamide gel under reducing conditions. Proteins were transferred onto nitrocellulose membranes. Membranes were blocked with 5% (wt/vol) skimmed milk powder in phosphate-buffered NaCl-0.1% Tween 20 overnight at 4°C and incubated with primary antibodies diluted in blocking buffer for 1 h at room temperature. After five washes in blocking buffer, membranes were incubated with alkaline phosphatase-conjugated secondary antibody diluted in blocking buffer for 30 min at room temperature. Membranes were finally washed five times in blocking buffer and twice in 0.1 M Tris, pH 9.5, containing 0.05 MgCl2 and 0.1 M NaCl. Specific binding was detected by using nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl-phosphate (p-toluidine salt; Pierce) as substrates and quantitated by densitometry. Protein determination was done with the bicinchoninic acid system (Pierce) with bicinchoninic acid used as a standard. Antibodies against collagen types I (Ab 749) and II (Ab 746) and antibodies against proteoglycans (MAb 2015; recognizing short peptides substituted with keratan sulfate side chains and core protein in skin and cartilage proteoglycans) were purchased from Chemicon (Temecula, CA).

Real-time RT-PCR. Total RNA was extracted with RNeasy Mini columns (Qiagen, Hilden, Germany). Skin slices (~10–20 mg) were homogenized in 500 µl of RLT buffer reagent with an Ultra-Turrax for 30 s. After homogenization, 950 µl of water and 320 µg of proteinase K were added, and the sample was incubated at 55°C for 10 min and then centrifuged at 12,000 rpm for 3 min. After the addition of 1 ml of 96–100% ethanol, the solvent was transferred to the columns and eluted according to the standard protocol. First-strand cDNA was synthesized with TaqMan RT reagents (Applied Biosystems), with random hexamers used as primers. Final RNA concentration in the reaction mixture was adjusted to 0.1 ng/µl. Reactions without Multiscribe reverse transcriptase were used as negative controls for genomic DNA contamination. RT products were diluted 1:1 with distilled H2O before the PCR procedure. PCR was performed with an ABI PRISM 7000 sequence detector and SYBR green reagents (Applied Biosystems) according to the manufacturer's instructions. Primers used for amplification are shown in Table 1. All samples were run in duplicate. The relative amount of the specific mRNA of interest was normalized with respect to 18S rRNA. Dissociation curves were performed to confirm the specificity of the PCR.


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  		Table 1. Real-time PCR primers

 
Data analysis. We estimated osmotically inactive skin Na+ storage relative to ECF. On the assumption that osmotically inactive Na+ reservoirs were emptied in rats fed a 0.1% NaCl diet for 8 consecutive weeks, ECF (ECF0.1%) and intracellular fluid (ICF0.1%) were calculated from the ratio of skin Na and skin water [R(SkNa+/SkW)] to serum Na+ concentration ([Na+]serum) in rats fed 0.1% NaCl

(1)
and

(2)
Assuming that water accumulation in rats fed a high-NaCl diet was ECF, while ICF remained unchanged, we used the water accumulation relative to wet skin weight ({Delta}rSkW, ml/g WW) from a low- to a high-NaCl diet and absolute skin wet weight (WW, g) and SkW (ml) to calculate ICF in rats fed an 8% NaCl diet (ICF8%)

(3)
and

(4)
The skin Na+ concentration in rats fed 8% NaCl ([SkNa+]8%, mmol/ml) was estimated from R(SkNa+/SkW) relative to the ECF

(5)
Finally, the fraction of osmotically inactive Na+ (Nai+) relative to ECF was

(6)

Differences in rSkW, R(SkNa+/SkW), and [Na+]serum were analyzed with multivariate analysis (GLM). mRNA data from rat skin were analyzed with the nonparametric Mann-Whitney test for independent samples. Statistics were calculated with SPSS software (version 10.0).


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Osmotically inactive Na+ storage in skin. From R(SkNa+/SkW) and Na+ (mmol) and water (ml) content in skin of animals fed different NaCl diets, we indirectly estimated Nai+ relative to ECF from the data presented in Table 2. On the basis of the assumption that there was no osmotically inactive skin Na+ storage in rats fed a low-NaCl diet, ECF in skin of rats fed 0.1% NaCl was 0.101 mmol Na+/ml SkW/0.155 mmol Na+/ml ECF = 0.652 ml ECF/ml SkW (Eq. 1) and ICF was 1 – 0.652 = 0.348 ml ICF/ml SkW (Eq. 2). In rats fed a high-NaCl diet, ICF was (25.6 – 0.053 x 44.5) x 0.348 ml ICF/ml SkW/25.6 = 0.316 ml ICF/ml SkW (Eq. 3) and ECF was 1 – 0.316 = 0.684 ml ECF/ml SkW (Eq. 4). The range of these estimations corresponded well to ECF determination in rat skin on the basis of isotope equilibration techniques (19). Thus the Na+ concentration relative to extracellular skin water was 0.128 mmol/0.684 ml = 0.187 mmol/ml (Eq. 5) in rats fed 8% NaCl. As judged from [Na+]serum, osmotically inactive skin Na+ storage was 0.187 – 0.152 = 0.035 mmol/ml ECF (Eq. 6) in the rats fed 8% NaCl (Fig. 1).


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  		Table 2. Skin wet weight, skin water, relative skin water, skin Na+-to-skin water ratio, and serum Na+ concentration

 


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  		Fig. 1. Estimated extracellular skin Na+ concentration in female Sprague-Dawley rats fed 0.1% or 8% NaCl for 8 consecutive wk. Na+(a), osmotically active Na+; Na+(i), osmotically inactive Na+; ECF, extracellular fluid.

 
Na+ storage and extracellular matrix proteins in cartilage and skin. Figure 2 shows Western blot analysis of extracellular matrix proteins collagen I/II, GAG-carrying proteoglycans, in three individual rats with low, medium, and high skin Na+ content relative to dry weight (rSkNa+, mmol/g dry wt). Densitometric units of the different lanes are shown in Table 3. Cartilage collagen II and cartilage proteoglycan content was higher with increasing rSkNa+ in the rats. In particular, increases in the small proteoglycan fragment content (lane 4, <110 kDa) in cartilage coincided with a high rSkNa+. Compared with cartilage, a direct correlation between rSkNa+ and collagen I expression in skin was less readily apparent in the rats. Similar to cartilage, small proteoglycan fragment content in skin (lane 4, <110 kDa) increased with increasing rSkNa+.



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  		Fig. 2. Western blot analysis of collagen I and II and proteoglycan content in cartilage and skin in 3 female Sprague-Dawley rats with low (+), medium (++), and high (+++) skin Na+ content. Control, skin and cartilage from a female rat before dietary NaCl loading or restriction.

 

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  		Table 3. Densitometric quantification of collagen I/II and proteoglycan content

 
Skin Na+ storage and expression of GAG chain polymerization enzymes in rats. Figure 3A shows the relative mRNA content of chondroitin synthase 1 (CSS) in the skin in rats fed a low- or a high-NaCl diet for 8 consecutive weeks. Osmotically inactive skin Na+ storage coincided with increased gene expression of CSS. The DS polymerization enzyme 4-N-acetylgalactosaminyltransferase (Fig. 3B) and 1,3-acetylgalactosaminyltransferase (Fig. 3C) showed gene expression similar to the CSS in rats fed a high-NaCl diet. In contrast to these key enzymes of GAG chain elongation, the GAG initiation enzyme xylosyl transferase 2 (Fig. 3D) mRNA content was unchanged in animals fed the high-NaCl diet. Similar to the initiation enzyme, mRNA expression of the small proteoglycans biglycan (Fig. 4A) and decorin (Fig. 4B) was not different between the dietary regimens.



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  		Fig. 3. Real-time PCR analysis of chondroitin synthase 1 (A), 4-N-acetylgalactosaminyltransferase (B), 1,3-acetylgalactosaminyltransferase (C), and xylosyl tranferase 2 (D) mRNA content in rat skin. Rats were fed 0.1% or 8% NaCl. *P < 0.05; tP = 0.05; ns, not significant. Boxes show 25th percentile, median, and 75th percentile; x shows maximum and minimum; {square} shows average.

 


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  		Fig. 4. Real-time PCR analysis of decorin (A) and biglycan (B) mRNA in rat skin. Rats were fed 0.1% or 8% NaCl. See Fig. 3 legend for explanation of format.

 

   DISCUSSION
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The important findings in our study were that osmotically inactive skin Na+ storage correlates with specific changes in GAG metabolism in adult female rats. It remains to be investigated whether these results are gender or age specific and whether these data are transferable to milder dietary salt loading protocols. As estimated from the relation between Na+ and water accumulation, "water-free" Na+ storage increased the theoretical extracellular skin Na+ concentration to ~180–190 mmol/l in rats fed 8% NaCl (Fig. 1). Increasing skin Na+ content in the rats was associated with increasing proteoglycan content in cartilage and skin (Fig. 2). RT-PCR analysis revealed an increased chondroitin synthase skin mRNA content in rats fed the high-NaCl diet (Fig. 3), whereas xylosyl transferase and small proteoglycan mRNA content was unchanged.

The role of GAGs in Na+ metabolism has been investigated previously. Farber et al. (3, 4) found that cartilage CS acts as a polyanion in the organism and concluded that "cations of mucoprotein and chondroitin sulfate behave as if they are incompletely ionized." He hypothesized that "excess sodium could exist in association with a polyelectrolyte as an osmotically inactive ion, a situation which would explain an abundance of sodium without equivalent amounts of water." In cartilage, chondrocytes are embedded in an extracellular matrix with a high polyanionic GAG content. Their extracellular ionic environment is thus different from that of most other cells. The extracellular Na+ concentration in cartilage is variable, i.e., 250–350 mmol/l ECF (17). Differences in the cartilage GAG concentration are directly correlated with corresponding Na+ changes in the extracellular matrix (11). Correspondingly, cartilage osmolality is 350–450 mosmol/kg. This osmotic strength maintains a high water content in cartilage, which provides its biomechanical properties. On the other hand, the high electrolyte concentrations indicate osmotically inactive Na+ storage in cartilage.

Some of these notions may be transferable to osmotically inactive skin Na+ storage. Total body Na+ and, thus, skin Na+ excess coincided with osmotically inactive Na+ storage and increasing GAG content in cartilage and skin. The GAG increase in rat skin and increasing mRNA levels of the DS elongation enzyme chondroitin synthase were associated with an extracellular Na+ concentration of ~180–190 mmol/l in the skin in vivo. Inasmuch as we cannot supply data for the skin GAG charge density in this experiment, we can only speculate that, similar to cartilage, increasing skin GAG content and, thus, an increasingly negative skin GAG tissue charge density might be the basic physiological mechanism responsible for osmotically inactive skin Na+ storage. The elongation of the DS chain could play a central role in osmotically inactive skin Na+ storage by increasing the negative charge density in the tissue.

We recently reported that an inbred (13) or an acquired (14) deficiency in osmotically inactive Na+ skin storage predisposed rats to a volume-sensitive blood pressure increase. We also observed that volume regulation only partially characterizes total body Na+ metabolism. Furthermore, natriuresis as the effector of Na+ homeostasis was not only regulated by the circulating volume, but also by the Na+ content in osmotically inactive Na+ reservoirs (14). Interestingly, the epithelial Na+ channel (ENaC) is expressed in articular cartilage (16) and skin (1, 8). ENaC expression on keratinocytes is related to cell differentiation and is required for normal epidermal growth (8). The physiological role of ENaC expression in articular cartilage is unknown. One may speculate that ENaC expression in chondrocytes, fibroblasts, or keratinocytes could play a role as an extracellular Na+-sensing mechanism that regulates osmotically inactive Na+ storage. From the data we present here, we speculate that osmotically inactive skin Na+ storage might be an active process that involves an increased GAG polymerization, increasing the negative charge density in skin that attracts Na+ ions to leave their completely hydrated state.

We believe that our findings have clinical implications. First, our observations could explain the discrepancies reported in various Na+ balance studies (6, 15). Second, our observations may have important implications for salt-sensitive hypertension. Finally, skin biopsies could possibly be applied to human subjects to estimate total body Na+ content or long-term Na+ intake.


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This work was supported by grants from the Else Kröner-Fresenius-Stiftung and the Deutsche Nierenstiftung to J. Titze. Costs of publication were defrayed by the ELANS Fonds, University Erlangen.


   ACKNOWLEDGMENTS
 
We thank B. Weigel and S. Böhm for help with the animal experiments and E. Prell for help with the ashing procedures. We thank F. C. Luft for continuous support and for correcting the manuscript.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. Titze, Nephrologische Forschungslaboratorien Medizinische Klinik IV, Loschgestrasse 8, D-91054 Erlangen, Germany (E-mail: jus.titze{at}t-online.de).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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