Background
Urinary Ca
2+ excretion increases with sodium chloride (NaCl) ingestion [
1]. This dietary NaCl-induced calciuria may lead to osteoporosis at low calcium intake [
2,
3] and also is associated with urinary stone formation [
1] and hypertension [
4]. The increase in urinary Ca
2+ excretion is postulated to be due to salt-induced volume expansion [
5] and/or competition between sodium and calcium ions in the renal tubule [
6]. However, the precise mechanism for the dietary NaCl-induced urinary Ca
2+ increase is not fully understood. In addition, it is not clear if long-term salt loading has any effects on Ca
2+-transporting molecule expressions in the kidney.
The bulk of Ca
2+ in the pro-urine is reabsorbed in the proximal tubule and the thick ascending loop of Henle through a passive, paracellular movement. Transepithelial Ca
2+ permeability is high in these segments, and the rate-limiting barrier is the tight junction. Claudins and other tight junction proteins are known to be important in determining the permeability characteristics of various epithelia [
7]. For example, renal expression of claudin 2 is restricted to the proximal nephron [
8], and claudin 2 is believed to form high-conductance cation pores [
9]. The distributions and functions of these tight junction proteins are becoming known, but information on their regulation, especially in the kidney, is just emerging.
In contrast, regulated transcellular Ca
2+ reabsorption occurs primarily in the distal tubule. In the distal nephron, Ca
2+ in the pro-urine enters the cytosol of tubule cells through Ca
2+ channel, mainly TRPV5 [
10]. The transport of intracellular Ca
2+ to the basolateral side is facilitated by a Ca
2+-binding protein called calbindin-D
28k[
10,
11], and Ca
2+ exits the cell on the basolateral side through Na
+/Ca
2+ exchanger 1 (NCX1) and Ca
2+ pump (PMCA1b) [
12,
13]. NCX1 counter-transports 3 Na
+ for Ca
2+, but the role of NCX1 in NaCl-induced calciuria has not been studied.
Alterations in the expressions of tight junction proteins and transcellular Ca2+ transporters may in part explain the urinary calcium loss upon salt loading or provide clues on long-term effects of dietary NaCl ingestion. Therefore, we examined the expression changes of renal Ca2+ transport molecules in rat with chronic high-NaCl diet.
Discussion
This study, for the first time, examined the effects of long-term dietary sodium chloride on renal Ca2+-transporting molecule and claudin expressions. Chronic salt loading decreased the protein expression of claudin 2, a component of proximal, paracellular Ca2+ transport pathway. Concomitantly, dietary NaCl increased the expression of more distal, transcellular Ca2+ reabsorption machinery, TRPV5, calbindin-D28k and NCX1.
Salt loading acutely increases urinary excretion of Ca
2+ along with Na
+[
3,
6]. In this study, the fractional Ca
2+ excretion of salt-loaded rats increased approximately 6.0 fold. Generally, the cause for this phenomenon is attributed to an extracellular fluid volume expansion and/or to the reduced reabsorption of both Na
+ and Ca
2+ in the proximal tubule [
36]. Although renal blood flow is reported to be unchanged or sometimes even reduced when salt loading is chronic such as over 8 weeks [
37], from this study, the contribution of volume expansion and/or hyperfiltration cannot be ruled out as creatinine clearance tended to increase in the salt-loaded rats, although not significant. As creatinine determination in rodents can vary depending on the method used [
38], the use of inulin clearance may be favorable. Pressure natriuresis is another possible factor of salt-induced calciuria, as blood pressure of salt-loaded rats tended to increase, although the difference was not statistically significant. Renal artery servo-control experiments would be useful to delineate these in the future.
Approximately 65% of calcium in the pro-urine is reabsorbed in the proximal tubule. In the proximal tubule, claudin-2 is postulated to form tight junction cation pores [
9]. Muto et al. have reported that fractional excretion of Ca
2+ in claudin-2 knockout mice is 3 times that of wild-type mice, further supporting a role of claudin-2 in proximal tubular paracellular Ca
2+ reabsorption [
39]. In this study, we found that chronic salt loading decreased renal cortical claudin-2 protein expression. Although there is not enough functional studies of rat claudin-2, sequence similarity to mouse claudin-2 suggests a similar role in Na
+ and Ca
2+ transport. Therefore, the decreased expression of claudin-2 with high-salt diet may, to some degree, account for the decrease in Ca
2+ reabsorption, while limiting Na
+ and water reabsorption, as Na
+[
9] and water [
40] in addition to Ca
2+ may pass through the pores formed by claudin-2. It has been reported that hyperosmolarity stress decreased claudin-2 expression in Madin-Darby canine kidney cells [
41], and hyperosmolarity due to NaCl load may be a possible mechanism of claudin-2 downregulation in this study. As claudin-2 facilitates Ca
2+ movement from the luminal to interstitial fluid in the proximal tubule, reduction in claudin-2 may underlie the increased urinary Ca
2+ excretion observed under high-salt diet.
In the proximal tubule, NHE3 is shown to be important as a part of driving force for Ca
2+ reabsorption, mediating apical Na
+ entry and consequently water reabsorption to produce osmotic gradient [
18]. In our study, renal NHE3 protein significantly increased with salt loading. However, this finding is not in accordance with some previous studies, such as that of Frindt and Palmer who found no change in luminal NHE3 with 5% NaCl diet for 1 week in rats using in situ biotinylation [
42]. As regulation of NHE3 occurs on multiple levels, including trafficking, interacting proteins and oligomerization [
43], protein level may not be directly related to apical NHE3 activity. If NHE3 activity is indeed increased in the high salt-fed rats, this may increase the pressure for Ca
2+ reabsorption in the proximal tubule. However, competition between Na
+ and Ca
2+ for paracellular transport binding site may occur in the proximal tubule. It has been reported that Ca
2+ inhibits paracellular Na
+ conductance by competitive binding on claudin-2 [
44]. If Na
+ and Ca
2+ share a binding site, inversely, high Na
+ may inhibit claudin-2 Ca
2+ conductance. This competition between Na
+ and Ca
2+ may play a large role in the dietary NaCl-induced hypercalciuria.
Thick ascending limb of the loop of Henle is responsible for approximately 20% of Ca
2+ reabsorption. Claudin-16 and −19 are shown to be important for paracellular Mg
2+ and Ca
2+ in this segment. In our study, there was an increase in the fractional excretion of Mg, albeit smaller than that of Ca. However, there was no significant difference in renal claudin-16 or −19 mRNA in rats on high-salt diet. Extracellular volume expansion decreases transepithelial voltage and Mg
2+ reabsorption in the TAL [
45]. Although not directly detectable in our experimental setting, there may have been some volume expansion in the high-salt fed rats which may have contributed to the increase in Mg
2+ fractional excretion.
Distal nephron is the final and most-regulated site of urinary Ca
2+ reabsorption [
46,
47]. A concerted increase in the expression levels of TRPV5, calbindin-D
28k, and NCX1, was observed with salt loading in this study. Claudin-8, the distal tubular paracellular cation barrier, was not altered by salt loading. It may be that with salt loading, the proximal, paracellular Ca
2+ reabsorption is reduced, and more distal, transcellular Ca
2+ transport molecules are upregulated to facilitate Ca
2+ reabsorption as a compensatory mechanism. However, salt loading may reduce the Ca
2+ reabsorption via NCX1, as illustrated in Figure
7. Therefore, the upregulation of distal Ca
2+ transport machinery with chronic salt-loading may partially compensate for the urinary Ca
2+ loss, although with a limited effect.
As for the mechanism of TRPV5, calbindin-D
28k, and NCX1 upregulations by dietary NaCl, one possibility is the endocrine factors that regulate Ca
2+-related molecules, such as parathyroid hormone [
48] and vitamin D [
49]. For example, 1,25(OH)
2D has been shown to increase the expressions of TRPV5, calbindinD
28k, and NCX1 [
35]. However, in this study, serum concentration of 1,25(OH)
2D was significantly lower in the high-salt group than the control group. Unless there is a significant difference between serum and intrarenal 1,25(OH)
2D levels, it is likely that salt-induced transcellular Ca
2+-transporter upregulation is mediated by pathway(s) other than 1,25(OH)
2D.
The weakness of the study includes a lack of regional expression data, as excised renal cortex was used in the study. Higher-resolution immunohistological staining experiments and qRT-PCR/Western blotting from micro-dissected tissue specimens are necessary in the future. However, this study aimed to lay the foundation for a more detailed mechanistic examination of the effects of chronically high dietary sodium on the expression of renal Ca transporters and on urinary calcium excretion.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
MSY conceived the experiments in part with JY. KT, YM, RS and SA performed the experiments. HS, JK and TW gave advice on the study. All authors read and approved the final manuscript.