Calcium Extrusion Pump PMCA4: A New Player in Renal Calcium Handling?

Calcium (Ca2+) is vital for multiple processes in the body, and maintenance of the electrolyte concentration is required for everyday physiological function. In the kidney, and more specifically, in the late distal convoluted tubule and connecting tubule, the fine-tuning of Ca2+ reabsorption from the pro-urine takes place. Here, Ca2+ enters the epithelial cell via the transient receptor potential vanilloid receptor type 5 (TRPV5) channel, diffuses to the basolateral side bound to calbindin-D28k and is extruded to the blood compartment via the Na+/Ca2+ exchanger 1 (NCX1) and the plasma membrane Ca2+ ATPase (PMCA). Traditionally, PMCA1 was considered to be the primary Ca2+ pump in this process. However, in recent studies TRPV5-expressing tubules were shown to highly express PMCA4. Therefore, PMCA4 may have a predominant role in renal Ca2+ handling. This study aimed to elucidate the role of PMCA4 in Ca2+ homeostasis by characterizing the Ca2+ balance, and renal and duodenal Ca2+-related gene expression in PMCA4 knockout mice. The daily water intake of PMCA4 knockout mice was significantly lower compared to wild type littermates. There was no significant difference in serum Ca2+ level or urinary Ca2+ excretion between groups. In addition, renal and duodenal mRNA expression levels of Ca2+-related genes, including TRPV5, TRPV6, calbindin-D28k, calbindin-D9k, NCX1 and PMCA1 were similar in wild type and knockout mice. Serum FGF23 levels were significantly increased in PMCA4 knockout mice. In conclusion, PMCA4 has no discernible role in normal renal Ca2+ handling as no urinary Ca2+ wasting was observed. Further investigation of the exact role of PMCA4 in the distal convoluted tubule and connecting tubule is required.

Introduction

Calcium (Ca2+) is involved in several important processes in the body, including muscle contraction, bone mineralization and as a second messenger in multiple signal transduction pathways [1]. As a consequence, the plasma Ca2+ concentration is tightly controlled via absorption of dietary Ca2+ at the intestine, storage in bone and reabsorption by the kidney [2, 3]. In the kidney the majority of filtered Ca2+ is passively reabsorbed in the proximal part of the nephron, though fine-tuning occurs in the late distal convoluted tubule (DCT) and the connecting tubule (CNT) [4]. This is an active process where Ca2+ reabsorption can be regulated by hormones including parathyroid hormone (PTH) and active vitamin D (1,25(OH)2D3) [5-7]. In the late DCT and CNT Ca2+ enters the cell from the pro-urine via the apically expressed transient receptor potential vanilloid channel type 5 (TRPV5) [8, 9]. Subsequently, Ca2+ binds to calbindin-D28k (CaBP28k) and/or calbindin-D9k (CaBP9k) and this complex diffuses to the basolateral side, where Ca2+ is extruded via the Na+/Ca2+ exchanger (NCX1) or plasma membrane Ca2+ ATPase (PMCA) [10, 11].

PMCA is a member of the P-type ATPase family, and is related to the sarcoplasmatic/endoplasmatic reticulum Ca2+ ATPase pumps. Four different genes encode for PMCA 1 to 4. Both PMCA1 and 4 are ubiquitously expressed and have been suggested to have a housekeeping function, extruding Ca2+ from the cell [12]. PMCA1 knockout (KO) mice are embryonically lethal, whereas the PMCA4 KO mice are viable and appear healthy [13, 14]. This suggests that PMCA4 might have a more specific role than PMCA1. Indeed, PMCA4 has been shown to play a role in Ca2+ signaling in sperm motility, B-lymphocytes and cardiac nitric oxide signaling [13-16].

In the kidney, PMCA1 and 4 transcripts and protein are found in the proximal tubule, but higher expression has been shown at the distal part of the nephron [17, 18]. PMCA1 is considered as the predominant PMCA responsible for transcellular Ca2+ transport in the late DCT and CNT [18, 19]. Recently however, Alexander et al. investigated the exact localization of PMCA4 in the kidney, by co-staining the different tubular segments with representative markers [20]. They verified that PMCA4 was expressed highest in tubules that also expressed TRPV5. In addition, PMCA4 was decreased in TRPV5 KO mice, as were NCX1 and CaBP28k [21, 22]. On the contrary, PMCA1 was not changed [22]. This suggests that PMCA4 may be the predominant PMCA form involved in distal transcellular Ca2+ reabsorption.

In this study we assessed the role of PMCA4 in renal Ca2+ handling. To this end, the PMCA4 KO mouse model was used and its Ca2+ balance was compared to wild type (WT) and heterozygous (HZ) littermates. There were no significant differences in serum Ca2+ levels or urinary Ca2+ excretion between groups or changes in expression of Ca2+-related genes.

Material and Methods

Ethics statement

This study was carried out in strict compliance with the United Kingdom Animals (Scientific Procedures) Act 1986. All experimental procedures were approved by the University of Manchester Ethics Committee (permit-no: 40/3625) and all efforts were made to minimize suffering of animals. A completed ARRIVE guidelines checklist is included in S1 Checklist.

Animals

PMCA4 germline null mutant mice, as previously described [14], were maintained in a pathogen-free facility, housed under a 12 hour light/dark cycle, with ad libitum access to food and water. Experiments were performed with male WT (n = 10), HZ (n = 7) and KO (n = 10) mice when animals were aged 27–31 weeks old.

Immunofluorescence

Immunofluorescence staining was used to detect PMCA4 protein expression in kidney sections, and was performed as described previously [23]. In short, PMCA4 staining was performed on 5 μm thick sections of 1% (w/v) periodate-lysine-paraformaldehyde-fixed mouse kidney samples. Sections were incubated overnight at 4°C with mouse anti-PMCA4 (1:100, ab2783, Abcam, Cambridge, UK) and incubated with Alexa Fluor 488 conjugated secondary antibody (1:300, Invitrogen, Carlsbad, CA, USA) for 2 hours. Images were collected with an AxioCam camera and AxioVision software (Zeiss, Sliedrecht, The Netherlands).

Metabolic cage study

To collect urine, mice were housed individually in metabolic cages for 24 hours (from 10am to 10am) with ad libitum access to food and drinking water. The amount of water and food provided to each mouse was recorded at the start and after the 24-hour period, to determine amounts ingested. Following the metabolic cage housing, blood was collected via the jugular vein under isoflurane anesthesia and stored overnight at 4°C. All animals were humanely sacrificed by cervical dislocation. Kidneys were isolated, decapsulated, subdivided and snap frozen in liquid nitrogen. Duodenum segments were extracted, cleaned with PBS and snap frozen in liquid nitrogen. All samples were stored at -80°C until required. Serum was obtained following centrifugation of whole blood samples at 500g for 10 min, and frozen at -80°C until required. One KO mouse presented with fluid filled kidney cysts, and was not included in data analysis.

Analytical procedures

Urinary osmolality was measured using an Advanced® Model 3320 Micro-Osmometer (Advanced instruments Inc., Norwood, MA, USA). A colorimetric assay was used to determine serum and urine Ca2+ concentrations as described previously [22]. Ca2+ measurements were verified using an internal control, which was a commercial serum standard (Precinorm U, Roche, Basel, Switzerland). Serum and urinary phosphate (Pi) concentrations were determined by in-hospital services using automatic biochemical analyzers. Serum PTH levels were determined using the mouse PTH 1–84 ELISA kit (Immunotopics international, San Clemente, CA, USA) and serum Fibroblast Growth Factor (FGF)23 levels were determined with the mouse C-terminal FGF23 ELISA assay (Immunotopics International).

Quantitative real-time PCR

Total RNA was isolated from kidneys and duodenum using TRIzol® reagent (Invitrogen) according to the manufacturer’s protocol and dissolved in 150 μl diethylpyrocarbonate (DEPC)-treated deionized water, and stored at -80°C. Thereafter, RNA was treated with RQ1 DNase (1U, Promega, Madison, WI, USA) and reverse transcription was performed using 1.5 μg RNA and Moloney murine leukemia virus reverse transcriptase (200 U, Invitrogen) for 1.5 hours following the manufacturer’s protocol, except that RNasin® (Promega) was used as an RNase inhibitor [22]. The obtained cDNA was used to determine mRNA levels of several Ca2+-related genes, as well as glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as an endogenous control [22]. All the primers used (Biolegio, Nijmegen, The Netherlands) are listed in S1 Table. SYBR® Green mastermix (Bio-Rad, Veenendaal, The Netherlands) was used to perform quantitative real-time PCR according to manufacturer’s protocol and samples were measured on a CFX96 Bio-Rad analyzer (Bio-Rad). Gene expression was normalized for Gapdh expression and calculated according to the ΔΔCT method [24]. The PMCA4 real-time PCR product from WT, HZ and KO samples were run on a 2% (w/v) agarose gel electrophoresis.

Protein isolation from kidney and immunoblotting

Total kidney protein lysates were prepared as described previously [25] and 30 μg protein was loaded to either 8% (w/v) or 12% (w/v) SDS-PAGE gel for NCX1 and CaBP28k, respectively and blotted to a PVDF membrane (Millipore, Billerica, MA, USA). Immunoblots were incubated overnight at 4°C either with mouse anti-CaBP28k (1:5,000, clone CB-955, Sigma-Aldrich, Zwijndrecht, The Netherlands), mouse anti-NCX1 (1:500, ab6495, Abcam) or mouse anti-beta-actin (1:10,000, clone AC-15, Sigma-Aldrich). Immunoblots were enhanced using chemiluminescence (ECL, Pierce, Etten-Leur, The Netherlands) and analyzed using a ChemiDoc XRS (Bio-Rad) system. Semi-quantification was performed as described previously [26].

Statistics

Values are expressed as mean ± S.E.M. Differences between the WT, HZ and PMCA4 KO mice were tested using a one-way ANOVA with Tukey post-hoc test. Differences were considered significantly different when P<0.05. Analysis of the dataset was performed using GraphPad Prism, version 6.0.

Results

Similar serum Ca2+ levels and urinary Ca2+ excretion in WT and PMCA4 KO mice

The role of PMCA4 in renal Ca2+ handling was investigated using PMCA4 WT, HZ and KO mice. By quantitative real-time PCR and immunofluorescence staining of the kidney cortex, PMCA4 mRNA and protein were not detected in kidneys from PMCA4 KO mice (Fig 1). Before the start of the animal experiment mice of the different groups were of similar body weight (data not shown). Subsequently, mice were housed in metabolic cages to collect 24-hour urine. There was no significant difference in body weight, kidney weight, food intake, diuresis or osmolality between the different groups (Table 1). Water intake was significantly decreased between WT and KO (Table 1). To examine if PMCA4 KO mice develop disturbances in their Ca2+ homeostasis, serum Ca2+ levels and 24-hour urinary Ca2+ excretion were measured; however, both were similar in all three groups (Fig 2).

Fig 1

Confirmation of PMCA4 knockout in the kidney.

(A) Renal relative mRNA expression levels (corrected for GAPDH) of PMCA4 in wild type (WT, n = 10), heterozygous (HZ, n = 7) and knockout (KO, n = 10) mice. Data represents mean ± S.E.M. *P<0.05 compared to WT. Real-time PCR products were subjected to 2% (w/v) agarose gel electrophoresis. (B-D) Representative immunofluorescence image for PMCA4 expression in mouse kidney cortex of wild type (B) and PMCA4 knockout mice (C). (D) Negative control without the primary antibody.

Table 1

Characteristics of PMCA4 wild type, heterozygous and knockout mice.

	Wild type (n = 10)	Heterozygous (n = 7)	Knockout (n = 10)
Body weight (g)	27.0±0.4	28.7±0.7	28.3±0.9
Kidney weight (g)	0.36±0.01	0.38±0.01	0.37±0.01
Food intake (g/24h)	2.1±0.2	2.5±0.4	2.0±0.2
Water intake (mL/24h)	3.3±0.4	3.1±0.5	1.9 ±0.4*
Diuresis (mL/24h)	2.1±0.3	2.2±0.5	1.5±0.3
Osmolality (mOsm/kg)	1458±151	1644±299	2099±236

Wild type, heterozygous and knockout mice were housed for 24 hours in metabolic cages, after which body and kidney weight, food and water intake, diuresis and osmolality were measured. Data represents mean ± S.E.M.

*P<0.05 compared to wild type.

Fig 2

No difference in serum and urinary Ca2+ levels between the different PMCA4 genotypes.

Serum Ca2+ (A) and 24-hour urinary Ca2+ excretion (B) in wild type (WT, n = 10), heterozygous (HZ, n = 7) and PMCA4 knockout (KO, n = 10) mice. Data represents mean ± S.E.M.

No renal or duodenal compensation of Ca2+-related genes in PMCA4 KO mice

Renal mRNA expression of Ca2+-related genes was analyzed to determine possible compensation mechanisms following PMCA4 ablation. Expression of the epithelial Ca2+ channel TRPV5, the so-called Ca2+ gatekeeper of the late DCT and CNT [27], was not significantly different between groups (Fig 3A). In addition, other genes involved in Ca2+ reabsorption in the late DCT and CNT, including NCX1, PMCA1, CaBP28k and CaBP9k, were similar between the genotypes (Fig 3B–3E). In addition, renal protein expression of CaBP28k and NCX1 was not significantly different between groups (Fig 3F and 3G). Besides kidney, duodenal mRNA expression of Ca2+-related genes was measured to establish if potential compensation occurred in the duodenum. No difference was found for TRPV6, the active Ca2+ absorption channel in the duodenum [28] (Fig 4A). Moreover, NCX1, PMCA1 and CaBP9k were comparable between the three genotypes (Fig 4B–4D).

Fig 3

No effect of PMCA4 ablation on mRNA or protein levels of Ca2+-related genes in the kidney.

Relative mRNA expression levels (corrected for GAPDH) of TRPV5 (A), NCX1 (B), PMCA1 (C), calbindin-D28k (CaBP28k, D) and calbindin-D9k (CaBP9k, E) were determined in kidneys of wild type (WT, n = 10), heterozygous (HZ, n = 7) and PMCA4 knockout mice (KO, n = 10). (F) Representative immunoblot of renal CaBP28k and NCX1 protein expression in the three groups. Beta-actin was used as loading control. (G) Semi-quantification of immunoblots of CaBP28k and NCX1, corrected for beta-actin and compared to WT. Data represents mean ± S.E.M.

Fig 4

No effect of PMCA4 ablation on mRNA expression of Ca2+-related genes in duodenum.

TRPV6 (A), NCX1 (B), PMCA1 (C) and calbindin-D9k (CaBP9k, D) relative mRNA expression levels (corrected for GAPDH) were analyzed in the duodenum of wild type (WT, n = 10), heterozygous (HZ, n = 7) and PMCA4 knockout mice (KO, n = 10). Data represents mean ± S.E.M.

PMCA4 KO mice show increased FGF23 levels

In order to investigate whether hormonal Ca2+ regulation is changed in PMCA4 KO mice, PTH was measured in serum. No significant difference was present between the three groups of mice (Fig 5A). Moreover, renal mRNA expression of vitamin D activating (Cyp27b1) and breakdown (Cyp24a1) enzymes was investigated, and their expression was found not to significantly differ between genotypes (Fig 5B and 5C). Serum FGF23 levels were, however, significantly higher in PMCA4 KO mice compared to WT (Fig 4D). Since FGF23 is involved in regulating Pi homeostasis, serum Pi and 24-hour urinary Pi excretion were determined. There were, however, no significant differences between groups (S1 Fig). In addition, renal expression of klotho and Na+/Pi co-transporters NaPi-IIa and NaPi-IIc were determined, though also here no significant differences were observed (S1 Fig).

Fig 5

PMCA4 ablation does not affect serum PTH but increases FGF23 serum levels (A) Serum PTH levels were measured in wild type (WT, n = 10), heterozygous (HZ, n = 7) and PMCA4 knockout mice (KO, n = 10). Relative mRNA expression levels (corrected for GAPDH) of the renal vitamin D activating enzyme Cyp27b1 (B) and vitamin D degrading enzyme Cyp24a1 (C). (D) Serum FGF23 levels were significantly higher in KO mice compared to WT. Data represents mean ± S.E.M, *P<0.05 compared to WT.

Discussion

Importantly, for the first time, this study aimed to characterize the renal role of PMCA4. PMCA4 has recently been localized in Ca2+ transporting epithelial cells, promoting speculation about its functional role in transcellular Ca2+ transport at the distal nephron [20, 22]. In this study we show that ablation of PMCA4 has no significant effect on serum Ca2+ level or on renal Ca2+ excretion. Moreover, PMCA4 KO mice did not show Ca2+-related mRNA compensation in the kidney or duodenum.

The Ca2+ concentration in the body needs to be tightly regulated for multiple physiological processes [3, 29]. Disturbances in Ca2+ homeostasis can result in osteoporosis, heart failure or kidney stones [30, 31]. The late DCT and CNT are responsible for regulated active Ca2+ uptake, via the transcellular pathway. On the apical side, Ca2+ enters the cell via TRPV5, whereas NCX1 and PMCA1 and/or 4, located basolaterally, are responsible for Ca2+ extrusion [10,11, 22]. It was considered that PMCA1 was responsible for late DCT and CNT Ca2+ extrusion, since in the duodenum it is the principal Ca2+ efflux mechanism [19, 32]. However, Van der Hagen et al. showed that there was relatively more mRNA expression of PMCA4 in the late DCT and CNT compared to total kidney, whereas PMCA1 was not enriched in this segment [22]. This was confirmed at the protein level, where the most intense expression of PMCA4 in the kidney was observed in TRPV5 expressing tubules [20, 22]. In addition, the TRPV5 KO mouse showed a decrease in mRNA levels of CaBP28k, NCX1 and PMCA4, but not PMCA1 [22]. Furthermore, parathyroidectomized rats, klotho KO mice and 1α-hydroxylase KO mice showed co-regulation of TRPV5, CaBP28k and NCX1, but not of PMCA1 [5, 6, 33]. Therefore, we hypothesized that PMCA4, rather than PMCA1, is the predominant pump for renal Ca2+ homeostasis in the late DCT and CNT. However, in this study the PMCA4 KO mice did not show an increase in urinary Ca2+ excretion. PMCA1 and/or NCX1 might have compensated for the ablation of PMCA4, normalizing serum Ca2+ and urinary Ca2+ excretion. However, there was no significant upregulation of NCX1 at gene and protein level, or increased mRNA expression for PMCA1 in PMCA4 KO mice. Total ablation of either NCX1 or PMCA1 is embryonic lethal in mice, hence their role in Ca2+ extrusion in late DCT and CNT cannot be determined by using a global KO [13, 34]. On the contrary, TRPV5 KO mice are viable but present with severe hypercalciuria [21, 35]. Mice with depletion of the Ca2+ shuttling protein CaBP28k show varying results concerning their Ca2+ excretion, with no difference or increased Ca2+ excretion compared to WT mice, depending on the study [27, 36–38]. In addition, Gkika et al. treated CaBP28k KO mice with a low and high Ca2+ diet (0.02% w/w or 2% w/w), and did not observe a disturbance in Ca2+ homeostasis, even though CaBP28k is involved in transcellular Ca2+ transport [27]. This suggests that we cannot necessarily rule out PMCA4 having a role in renal Ca2+ handling even though the PMCA4 KO mouse does not show a changed Ca2+ homeostasis.

At the intestinal level the PMCA4 KO mice did not show a difference in the Ca2+-regulating genes, indicating that no compensation occurred. Recently Alexander et al., have shown that PMCA4 is located in the smooth muscle layer of the intestine, and not in the enterocytes, where PMCA1 was more highly detected [20], suggesting that PMCA1, rather than PMCA4, is primarily involved in transcellular Ca2+ transport in the duodenum [20, 28, 39]. An earlier study has shown that PMCA4 KO mice presented with lower trabecular bone volume, lower bone mineral density and an increase in osteoclast surface area compared to WT mice [40]. Bone could be a source of Ca2+ to maintain a normal serum Ca2+ level; however, there was no indication of renal Ca2+ wasting in our mice studied here.

Ca2+ homeostasis is mainly regulated by PTH and 1,25(OH)2D3 in response to low serum Ca2+ levels [41]. The PMCA4 KO mice do not show differences in Cyp27b1 or Cyp24a1 mRNA, nor in serum PTH, compared to WT mice. This is consistent with our other observations where PTH- and 1,25(OH)2D3-regulated genes, such as TRPV5 and TRPV6, are not changed [5, 6, 42]. Moreover, PMCA4 KO mice presented with significantly lower water intake. However, since there were no changes in body weight or urinary excretion, this could have been caused by biological variation. In addition, we know of no reports indicating an involvement of PMCA4 in thirst and/or water homeostasis. Interestingly, FGF23 levels were increased in PMCA4 KO mice. In the kidney, FGF23 decreases NaPi-IIa and NaPi-IIc expression, resulting in increased urinary phosphate excretion [43]. In addition, it downregulates Cyp27b1 and increases Cyp24a1 expression [44]. These consequences were not observed in PMCA4 KO mice. This suggests that PMCA4 might be involved in FGF23 production and/or signaling, although more research is necessary to confirm this. Besides its role as general Ca2+ extruder, others have already shown the involvement of PMCA4 in signaling transducing pathways as well [45, 46].

In conclusion, PMCA4 does not seem to be vital for basal renal Ca2+ handling. However, the presence of PMCA4 in specific segments of the kidney points to the molecule having a different or additional role compared to PMCA1 and therefore, their exact roles should be investigated in more detail.

The ARRIVE checklist.

(PDF)

Click here for additional data file.

Metabolic cage data.

(XLSX)

Click here for additional data file.

No difference in serum Pi or 24-hour urinary Pi excretion between the different genotypes.

Serum Pi (A) and 24-hour urinary Pi excretion (B) in wild type (WT, n = 10), heterozygous (HZ, n = 7) and knockout (KO, n = 10) mice. Relative mRNA expression of NaPi-IIa (C), NaPi-IIc (D) and klotho (E) were determined in the kidney. Data represents mean ± S.E.M.

(TIF)

Click here for additional data file.

Primer sequences used for real-time PCR.

(DOCX)

Click here for additional data file.