R158Q and G212S, novel pathogenic compound heterozygous variants in SLC12A3 of Gitelman syndrome

Abstract

The dysfunction of Na⁺-Cl⁻ cotransporter (NCC) caused by mutations in solute carrier family12, member 3 gene (SLC12A3) primarily causes Gitelman syndrome (GS). In identifying the pathogenicity of R158Q and G212S variants of SLC12A3, we evaluated the pathogenicity by bioinformatic, expression, and localization analysis of two variants from a patient in our cohort. The prediction of mutant protein showed that p.R158Q and p.G212S could alter protein’s three-dimensional structure. Western blot showed a decrease of mutant Ncc. Immunofluorescence of the two mutations revealed a diffuse positive staining below the plasma membrane. Meanwhile, we conducted a compound heterozygous model—Ncc R156Q/G210S mice corresponding to human NCC R158Q/G212S. Ncc^R156Q/G210S mice clearly exhibited typical GS features, including hypokalemia, hypomagnesemia, and increased fractional excretion of K⁺ and Mg²⁺ with a normal blood pressure level, which made Ncc^R156Q/G210S mice an optimal mouse model for further study of GS. A dramatic decrease and abnormal localization of the mutant Ncc in distal convoluted tubules contributed to the phenotype. The hydrochlorothiazide test showed a loss of function of mutant Ncc in Ncc^R156Q/G210S mice. These findings indicated that R158Q and G212S variants of SLC12A3 were pathogenic variants of GS.

Keywords

Gitelman syndrome / mouse model / compound heterozygous / hypokalemia / Slc12a3

1 Introduction

Gitelman syndrome (GS) is an autosomal recessive, salt-losing tubulopathy caused by mutations in SLC12A3 [1]. GS is characterized by hypokalemia, hypomagnesemia, and hypocalciuria [2]. The prevalence of GS is approximately 1:40 000, and it is much higher in Asian populations (approximately 1:1000) [3]. To date, functional studies of NCC primarily focus on X.L. oocytes [4–8]. Only a few experiments have been conducted on mammalian cell lines and mouse [9]. Meanwhile, the main treatment for patients with GS is individualized lifelong oral potassium or magnesium supplementation or both [10]. However, the therapeutic effect is not ideal. Thus, further efforts are needed to explore better treatments for GS, and an ideal model is a prerequisite for research.

Several mouse models have been used to study the pathophysiology of GS. Ncc-deficient mice (Ncc null mice) exhibit hypocalciuria, hypomagnesemia, and compensated alkalosis. This model is only presented with subtle perturbations of sodium, fluid volume homeostasis, and altered renal handling of Mg²⁺ and Ca²⁺, which are partially consistent with the clinical manifestations of GS [11,12]. Yang et al. generated a nonsense Ncc Ser 707X knock-in mouse in 2010, which exhibited typical phenotypes of patients with GS, including hypokalemia, hypomagnesemia, hypocalciuria, hypotension, and hypokalemic metabolic alkalosis with renal K⁺ wasting [13]. Subsequently, an Ncc T58M (human T60M) knock-in mouse model was generated to evaluate the physiologic effects of NCC phosphorylation in vivo [14]. Notably, the Ncc T58M knock-in mouse successfully stimulated the phenotype of GS. T60M is common in patients with GS, particularly in Han Chinese [15]. Patients with p.(T60M) variant have an earlier age of onset and lower urinary calcium–creatinine ratio, which indicated that T60M was a special mutation in GS [16]. Recent research has found an earlier age of onset and lower urinary calcium–creatinine ratio in the homozygous group than heterozygous and compound heterozygous groups of patients with GS. Thus, Ser707X is a nonsense variant, and T60 is a phosphoacceptor site, which are special types of variants of SLC12A3 in human [17]. Meanwhile, the previously studied mouse models were homozygotes, but compound heterozygous variants were more common compared with homozygous variants in patients with GS, which accounted for 70% [18,19]. Furthermore, whether complex heterozygous variants cause GS-like phenotypes in mice remains to be confirmed. These results have driven us to confirm the pathogenicity of compound heterozygous variant and create a new mouse model to study GS in vivo. Thus, we created a novel mouse model of GS using a compound heterozygous variant from a patient with GS.

We have established a cohort of GS since 2016. We constructed a compound heterozygous mouse model, Ncc^R156Q/G210S mice, to evaluate the pathogenicity of R158Q and G212S found in a patient from our cohort [20]. We analyzed the effect of the mutation on protein structure and localization in vitro and localization and function in vivo to confirm the pathogenicity of R158Q and G212S and provide a better model to discover a new treatment strategy for GS.

2 Materials and methods

2.1 Ethical statement

This study was approved by the ethics committee of Shandong Provincial Hospital Affiliated to Shandong University (approval number LCYJ: No. 2019-147). The participant had signed the written informed consent before participation. The study was performed in accordance with the Declaration of Helsinki.

2.2 Bioinformatic analysis

We performed sequence alignment of NCC homologous proteins on 11 species to confirm the conservation of mutant positions. Online software such as Mutation Taster, Poly Phen-2, and PROVEN was used to determine the potential effects of the two variants on NCC function. Modeling of wild-type (WT) and mutant protein was achieved using SWISS-MODEL workspace, and PyMOL Viewer was used to visualize the effect of two variants on the protein configuration of NCC.

2.3 Plasmid construction and cell transfection

WT NCC was obtained from a human cDNA library by PCR [4]. WT NCC cDNA was subcloned into pcDNA3.1 (Invitrogen, Carlsbad, USA). The FLAG tag was inserted on N-terminal of WT NCC. All selected variants were created by using the Quick-Change Site-Directed Mutagenesis Kit (Beyotime, D0206) according to the manufacturer’s instructions. The primers were as follows: c.473G>A: forward 5′-GGCGTGATCCTCTACCTGCAGCTGCCCTGGATTACGGCC-3′, reverse 5′-GGCCGTAATCCAGGGCAGCTGCAGGTAGAGGATCACGCC-3′; c.634G>A: forward 5′-CCTCATCTCCCGGAGTCTGAGCCCAGAGCTTGGGGGCTC-3′, reverse 5′-GAGCCCCCAAGCTCTGGGCTCAGACTCCGGGAGATGAGG-3′. The sequences of the plasmids were confirmed by Sanger sequencing. All plasmids were transiently transfected into MDCT cells using Lipofectamine 3000 (L3000-015, Invitrogen, USA). Whole-cell lysates were collected for protein extraction 48 h after transfection.

2.4 Immunofluorescence

Forty-eight hours after transfection, cells were grown on glass coverslips, and cell culture dishes were fixed with 4% paraformaldehyde, permeabilized with 0.3% Triton X-100, and blocked for 30 min in 2% FBS. Immunostaining was accomplished with anti-FLAG (1: 200; Proteintech) or anti-Ncc (1:300, Millipore) overnight at 4 °C. Species-specific Alexa Fluor 555 secondary antibodies (Invitrogen, Waltham, MA) were used at 1:1000 at room temperature for 1 h. Cell nucleus was visualized by DAPI (4′,6-diamidino-2-phenylindole, blue). Protein localization was observed by fluorescence microscopy (Leica TCS SP8 MP, Germany). The fluorescence intensity was quantified by ImageJ.

2.5 Protein extraction and Western blotting

Ncc protein at the cell surface from MDCT cells was extracted using the Membrane and Cytosol Protein Extraction Kit (Beyotime, Shanghai, China) according to the manufacturer’s instructions. Proteins from the kidneys were lysed using RIPA buffer with proteinase 2 inhibitors (Invitrogen). The proteins were denatured, separated with 8% SDS-polyacrylamide electrophoresis, and transferred to polyvinylidene difluoride membranes (Merck Millipore, Darmstadt, Germany). After incubation for 1 h with 5% non-fat milk in TBST, the membranes were incubated overnight at 4 °C with anti-FLAG (1:1000, Proteintech), anti-Ncc (1:2000, Millipore), anti-TRPV6 (1:200, Alomone), anti-TRPV5 (1:200, Alomone), anti-TRPM6 (1:200, Alomone), and anti-β actin (1:5000, Proteintech) antibodies. Then the membranes were incubated with the corresponding secondary antibodies at a 1:5000 dilution for 1 h at room temperature. Immune complexes were detected using chemiluminescence.

2.6 Generation of Ncc R156Q and G210S knock-in mice

We commissioned GemPharmatech Co., Ltd., to generate Ncc R156Q and G210S knock-in mice. The targeting vector was prepared using the CRISPR/Cas9 technique. The plasmid was constructed to simultaneously express the single-stranded guide RNA Cas9 at a specific site of the mouse Slc12a3 gene in the donor plasmid carrying the Slc12a3 R156Q or G210S fragment. The two abovementioned plasmids were simultaneously injected into mouse fertilized eggs. The positive F0 generation mice were validated by PCR detection and sequencing. Chimeric males were bred with C57BL/6 females to produce heterozygous Ncc^R156Q/+/Ncc^G210S/+ mice (F1). Homozygous Ncc^R156Q/R156Q/Ncc^G210S/G210S mice (F2) were produced by mating Ncc^R156Q/+ or Ncc^G210S/+ mice (F1) with each other. Compound heterozygous Ncc^R156Q/G210S mice were produced by mating Ncc^R156Q/+ and Ncc^G210S/+ mice (F1) with each other. WT, Ncc^R156Q/+/Ncc^G210S/+, Ncc^R156Q/R156Q/Ncc^G210S/G210S, and Ncc^R156Q/G210S littermates (F2) were bred, and tail genomic DNA was applied for PCR genotyping. The mice were raised in a 12 h day and night cycle and fed with normal raw chow diet (Na⁺: 0.13% (w/w); K⁺: 0.36% (w/w); Ca²⁺: 0.5% (w/w)) and plain drinking water ad libitum for 12–16 weeks. Mice were euthanized via a sodium pentobarbital overdose at 14 weeks for further research. All animal experiments were approved by the Ethics Committee of Shandong Provincial Hospital Affiliated to Shandong University. All experiments were conducted in accordance with the institutional and national guidelines for the care and use of laboratory animals.

2.7 Blood and urine analysis and blood pressure (BP) measurement

Blood was drawn from the angular venous plexus under light isoflurane anesthesia. Mice were kept in metabolic cages for urine collection. Serum and urine creatinine (Cr), urea nitrogen, Ca²⁺, Mg²⁺, Na⁺, K⁺, and Cl⁻ levels were determined using automated methods (BS-830 chemistry analyzer; Mindray, Shenzhen, China). The BP level of restrained conscious mice was measured by using a programmable tail-cuff sphygmomanometer (BP-2010A, Softron, Beijing, China).

2.8 RNA isolation and real-time quantitative RT-PCR

Total RNA from mouse kidney tissue was isolated using a TRIzol reagent (Takara, Tokyo, Japan) following the manufacturer’s instructions. RT reaction (20 µL) included 1 µg of total RNA, oligo-dT primer, random 6-mers, and reverse transcriptase (RT, Takara). Real-time PCR was performed in LC480 (Roche, Mannheim, Germany) according to the manufacturer’s instructions. SYBR green (Takara) was used to detect the amplification of 20 µL of cDNA using the absolute quantitative ΔCt method. Each reaction consisted of 10 µL of SYBR green, 1 µL of cDNA sample, 1 µL of each primer (10 µmol/L), and 7 µL of distilled water. The thermal cycling conditions were as follows: 5 min at 95 °C followed by 40 cycles at 95 °C for 10 s, 60 °C for 10 s, and 72 °C for 10 s. The primers used for mouse Ncc, Trpv5, Trpv6, Trpm6, and β-actin gene RT-PCR were as follows: Ncc: forward 5′-CCGACTGGAGAGGCATTGATGG-3′, reverse 5′-TGGCAAGGTAGGAGATGGTGGT-3′; Trpv5: forward 5′-GAAACTTCTCAATTGGTGGGTCAG-3′, reverse 5′-TTTGCCGGAAGTCACAGTT-3′; Trpv6: forward 5′- GTCATGTACTTTGCCAGAGGA-3′, reverse 5′-TATAGAAGGCTGAAGCAAATCCCA-3′; Trpm6: forward 5′-AAAGCCATGCGAGTTATCAGC-3′, reverse 5′-CTTCACAATGAAAACCTGCCC-3′; β-actin: forward 5′-GGCTGTATTCCCCTCCATCG-3′, reverse 5′-CCAGTTGGTAACAATGCCATGT-3′.

2.9 Immunohistochemical technique

Five-micron-thick slides from mouse kidneys were dewaxed and rehydrated. Antigen recovery was performed by autoclaving at 97 °C for 20 min using an antigen recovery solution (citrate buffer 10 mmol/L, pH 6.0). The sections were allowed to cool at room temperature and then washed three times in phosphate-buffered saline (PBS) for 3 min each time. Endogenous peroxidase activity was blocked by immersing the sections in a 3% hydrogen peroxide blocker (Dako) for 10 min and washed with three kinds of PBS. After this initial processing step, the sections were incubated with an anti-Ncc primary antibody (1:200–1:500, Millipore) overnight at 4 °C. Then they were incubated with the ultrasensitive non-biotin HRP detection system for 1 h. Finally, the sections were counterstained with hematoxylin, dehydrated, and fixed. Protein localization was observed by TissueFAXS Histo (TissueGnostics, Austria).

2.10 Hydrochlorothiazide (HCTZ) treatment

HCTZ (12.5 mg/kg), an NCC inhibitor, was intraperitoneally administered to WT (n = 10), and Ncc^R156Q/G210S (n = 8) mouse littermates and urine samples 4 h after a single-dose treatment were collected for analysis.

3 Results

3.1 Clinical characterization

The patient was a 65-year-old man from a cohort of GS that we have previously reported [20], who presented with hypokalemia, hypomagnesemia, hypocalciuria, polyuria, high urine sodium, and metabolic alkalosis (Tab.1). His serum potassium level, usually 2–2.6 mmol/L, was difficult to correct to a normal range after 10-day treatment with an intravenous and oral potassium supplement. The patient had a normal plasma renin activity with normal BP and no abnormal physical signs. The serum potassium level was elevated to 3.0–3.5 mmol/L after treatment with spironolactone, potassium, and magnesium supplementation. Complete DNA sequencing of the SLC12A3 gene revealed novel compound heterozygous variants, namely, c.473G>A/p.R158Q and c.634G>A/p.G212S [21].

3.2 Potential influence on NCC protein

Two missense variants in exon 3 (c.473G>A/p.R158Q) and exon 5 (c.634G>A/p.G212S; Fig.1) which were previously reported by our group [20] were strongly predicted to be pathogenic using three web-based programs, including Mutation Taster, PolyPhen-2, and PROVEN.

Based on ACMG, the mutation of c.473G>A/p.R158Q in SLC12A3 was pathogenic. This variant had been reported in multiple patients with GS (PM3_Strong) [20,22,23]. This mutation had a low frequency in the normal population database (PM2_Supporting). It was also pathogenic as predicted by three web-based programs (PP3). The c.634G>A/p.G212S was uncertain significance. This variant had been reported in patients with GS (PM3), which was not included in the normal population database (PM2_Supporting) [20,24]. Moreover, it was pathogenic as predicted by three web-based programs (PP3).

Based on the sequencing alignment, the locations of R158 and G212 were highly conserved among all 11 species (Fig.1). Moreover, p.R158Q and p.G212S were predicted to alter protein’s three-dimensional structure by using the SWISS-MODEL workspace program (Fig.1). The variant at codon R158Q broke the alpha-helix structure, whereas G212S changed the conformation of the carbon–oxygen double bond, which may affect the modification of NCC protein or its interaction with other proteins and ions (Fig.1).

3.3 Effects of SLC12A3 variants on NCC expression and localization in vitro

We tested the surface expression levels of WT and mutant NCC in MDCT cells via Western blotting (Fig.1). The surface expression of R158Q-NCC and G212S-NCC decreased compared with WT-NCC (Fig.1). The immunofluorescence of NCC in MDCT cells showed a different staining pattern in WT-NCC-transfected cells, where an intracellular protein was detected on the cell surface, and in R158Q-NCC and G210S-NCC-transfected cells, in which intranuclear stain was primarily observed (Fig.1).

3.4 Phenotype of the Ncc R156Q/G210S knock-in mice

Ncc R156Q and G210S knock-in mice were fed normal rodent chow diet (Na⁺: 0.13% (w/w); K⁺: 0.36% (w/w); Ca²⁺: 0.5% (w/w)), and phenotypes were evaluated at the age of 12–16 weeks. As shown in Fig.2, no significant difference in body weight was observed among groups of mice, indicating that these two variants did not affect the body weight of mice (Fig.2). Compared with WT mice, Ncc^G210S/G210S mice had increased urine volume (Fig.2). Meanwhile, compared with WT mice, homozygous mice (Ncc^R156Q/R156Q and Ncc^G210S/G210S) and compound heterozygous mice (Ncc^R156Q/G210S) exhibited hypokalemia, hypomagnesemia, and increased fraction of K⁺ excretion in urine (Fig.2–2E). Moreover, compound heterozygous Ncc^R156Q/G210S mice exhibited increased fractional excretion of Mg²⁺ (FEMg% 11.5 ± 7.8) compared with WT mice, increased volume of urine (1580 ± 510 μL/day) compared with heterozygous Ncc^G210S/+ mice, and increased excretion of Mg²⁺ in urine (urine Mg²⁺ 4.73 ± 3.6 μmol/day) compared with heterozygous Ncc^R156Q/+ mice. BP, serum, and urine electrolytes were not significantly different (NS) between heterozygous Ncc^R156Q/+/Ncc^G210S/+ mice and WT mice (Tab.2). However, the excretion of calcium in urine was normal in Ncc^R156Q/R156Q, Ncc^G210S/G210S, and Ncc^R156Q/G210S mice, which was not consistent with GS.

3.5 Renal Ncc expression and localization in Ncc R156Q and G210S knock-in mice

Compared with WT mice, the expression of Ncc mRNA measured by quantitative RT-PCR in the kidney of Ncc^R156Q/G210S mice was significantly decreased. The same results were observed in Ncc^G210S/G210S and Ncc^R156Q/G210S mice. However, in Ncc^R156Q/+ and Ncc^G210S/+ mice, the expression of Ncc mRNA was not significantly different compared with WT mice (Fig.3). Meanwhile, the expression of Ncc protein was dramatically deceased in Ncc^R156Q/R156Q, Ncc^G210S/G210S, and Ncc^R156Q/G210S mice (Fig.3). Immunofluorescence and immunohistochemistry were performed to observe the localization of Ncc protein in the kidney of Ncc^R156Q/G210S mice. In WT mice, most of the WT-Ncc was located in the apical membrane of distal convoluted tubule (DCT) cells (Fig.3 and 3D). In heterozygous variants, Ncc was located in the apical membrane, and some of the Ncc was located in the cytoplasm. However, in homozygous and compound heterozygous variants, Ncc was primarily located in the cytoplasm (Fig.3 and 3D), which indicated that the Ncc had an abnormal localization in compound heterozygous variants similar to homozygous ones.

3.6 Sex effect on phenotype in NCC^R156Q/G210S mice

The phenotype of patients with GS correlated with sex; therefore, we also analyzed the effect of sex on the phenotype of Ncc^R156Q/G210S mice. As shown in Fig.3, no significant differences in serum K⁺, serum Mg²⁺, and fractional excretion of potassium were observed between male and female mice. The fractional excretion of magnesium was increased in male (P < 0.05) and female mice with no significant difference (Fig.4). Moreover, the 24 h excretion of calcium in urine showed a decreasing trend in male mice, which was not observed in female mice. The mutant Ncc localization showed no difference in NCC^R156Q/G210S mice between sexes (Fig.4).

3.7 Expression of the magnesium channel and epithelial calcium channels in male and female NCC^R156Q/G210S mice

Mg²⁺ reabsorption channels—transient receptor potential cation channel, subfamily M, member 6 (Trpm6)—were examined to explore the cause of hypomagnesemia and difference in 24 h excretion of calcium in urine between male and female mice. The mRNA expression of Tprm6 was decreased in male and female CH mice with no significant difference (Fig.5). Meanwhile, two major Ca²⁺ channels were examined. The expression of TRPV5 and TRPV6 was increased in male CH mice compared with male WT mice and female CH mice. Consistent with the results of quantitative RT-PCR, the protein abundance of TRPV5 and TRPV6 in male CH mice was significantly increased (Fig.5 and 5C).

3.8 Response of HCTZ in Ncc^R156Q/G210S mice

We treated WT and Ncc^R156Q/G210S mice with HCTZ, an NCC inhibitor, to determine whether Ncc function was reduced or absent in Ncc^R156Q/G210S mice. The excretion rate of Na⁺ and K⁺ was used as an index of Ncc function. The urine excretion of Na⁺ and K⁺ was markedly increased (P < 0.05) after a single dose of HCTZ treatment (12.5 mg/kg; intraperitoneally) in WT mice. Meanwhile, the volume of urine was remarkably increased (P < 0.05), and the excretion of Ca^{2 +} likely decreased in WT mice. However, the urine excretion of Na⁺, K⁺, and Ca²⁺ was not significantly affected, and the volume of urine was slightly increased in Ncc^R156Q/G210S mice, indicating that Ncc function was lost in Ncc^R156Q/G210S mice (Fig.6).

4 Discussion

In this study, we created a compound heterozygous mouse model Ncc R156Q/G210S knock-in mice for the first time to model a patient with GS caused by NCC R158Q and G212S variants. Ncc^R156Q/G210S mice clearly exhibited typical GS features, including hypokalemia, hypomagnesemia, and increased fractional excretion of K⁺ and Mg²⁺ with normal BP level. Ncc^R156Q/G210S mice exhibited a blunted response to thiazide, indicating that mutant Ncc function was dramatically diminished. A remarkable reduction of Ncc mRNA and protein and abnormal localization of Ncc in the kidneys of Ncc^R156Q/G210S mice implied the loss of function of mutant Ncc in vivo. Our study confirmed the pathogenicity of R158Q and G212S variants. Moreover, this novel model was consistent with the general clinical and genetic characteristics of GS, indicating its application potential for the further research of GS.

In our GS cohort, we identified a patient with two variants of the SLC12A3 gene. The results of several online prediction software and alterations in the three-dimensional structure of the protein indicated that these two variants may be pathogenic. Therefore, we conducted further experiments on these two variants. To date, functional studies of NCC primarily focus on X.L. oocytes [4–8]. Only a few experiments have been conducted on mammalian cell lines [9]. Five classes of NCC variants in GS have been proposed [18,19,25]. In our study, we evaluated WT and mutant NCC expression and localization of MDCT cells. The cell surface expression of R158Q-NCC and G212S-NCC decreased, and both of them did not reach the plasma membrane. These results indicated that these two variants might have a strong effect on NCC protein.

We constructed two-point mutant mice to confirm the pathogenicity of the two variants. In our previously reported cohort of patients with GS [20], 88% of patients were compound heterozygotes, which was consistent with a previous report that compound heterozygous variants were more common in patients with GS (almost 70%) [18,19]. Nevertheless, a complex heterozygous mouse model has not been reported. Thus, we created compound heterozygous Ncc R156Q/G210S mice corresponding to R158Q/G212S in human.

GS is due to the inactivation of NCC variants in human. Two groups of animal models were constructed: GS-Ncc null mice and single-point knock-in mice. However, both models had some imperfections. On the one hand, Ncc null mice cannot feature the phenotype of GS [11]. On the other hand, although Ncc Ser707X and Ncc T58M knock-in mice successfully exhibited typical phenotypes of patients with GS [13,14], Ser707X and T58M were special types of variants in SLC12A3. Ser707X was a nonsense mutation, and T58M was a phosphorylation site. Meanwhile, in other studies, only homozygous mice showed a phenotype, which was not consistent with the genetic characteristics of GS. These results have driven us to create a new mouse model.

In our study, we comprehensively evaluated the phenotypes of mice with different genotypes. Ncc^R156Q/+ and Ncc^G210S/+ mice were phenotypically normal, but Ncc^R156Q/G210S mice clearly exhibited typical GS features, including hypokalemia, hypomagnesemia, and increased fractional excretion of K⁺ and Mg²⁺ with normal BP level and serum creatinine concentration, which was consistent with a previous report, that is, carriers behaved as healthy populations. Meanwhile, homozygote Ncc^R156Q/R156Q and Ncc^G210S/G210S mice also showed hypokalemia and hypomagnesemia. Consistent with experiments in vitro, mRNA and protein were decreased markedly in Ncc^R156Q/R156Q and Ncc^G210S/G210S mice, indicating that R156Q and G210S variants of Slc12a3 might interfere with transcription, and thereby affecting the protein expression of Ncc in vivo. The causes of the decrease in Ncc mutant mRNA abundance might vary. First, microRNAs could be influenced. MicroRNAs are a set of small, endogenous, highly conserved, non-coding RNAs that control the expression of about 30% genes at post-transcriptional levels. Typically, microRNAs impede the translation and stability of mRNAs (mRNA) [25]. Mutations in SLC12A3 could lead to the different combination of microRNA to SLC12A3, which could destroy the stability of target mRNA and inhibit the translation of target mRNA. Second, mRNA translation is blocked by the mutation of SLC12A3, which leads to mRNA decay [26]. The mode of cross-talk between translation and mRNA decay influences the regulation of protein output from a transcript, which is likely linked to the function of the encoded protein. Thus, the causes of the decrease in Ncc mutant mRNA abundance could be complicated, which remained to be explored. The same results were observed in Ncc^R156Q/G210S mice. Meanwhile, the sparse mutant Ncc was primarily located in the cytosol instead of the apical membrane of DCT cells in the kidney, which indicated that R158Q and G212S were type-I mutations.

Interestingly, hypocalciuria was not observed in our mouse model, which was not consistent with the phenotype of previously reported single-point mutant mice, in which hypocalciuria was evident [13,14]. However, hypocalciuria can be predictive in the clinical diagnosis of GS but not a clinical manifestation in all patients. Some patients with GS confirmed by DNA sequencing did not present with hypocalciuria, which was also proven in our cohort of GS [20,27,28]. The nature and severity of the biochemical abnormalities of GS are highly heterogeneous to intra-familial phenotype [29]. In addition, the fodder of mice was slightly different in composition from the ingredients used in previous literature. These reasons might explain the absence of hypocalciuria in mice that we constructed.

As previously reported, male might have a more severe phenotype than female [30]. In addition, the gender effect on phenotype in GS was supported by other studies [5,31]. Thus, we analyzed the phenotype in male and female Ncc^R156Q/G210S mice. We found a decreasing trend in male mice based on 24 h excretion of calcium in urine, which was not observed in female mice, but no statistical difference was observed. Calcium metabolism might be different between genders in our study through this analysis. The normal excretion of urinary calcium in female could be explained. Estrogen can increase calcium reabsorption in the kidney [32,33]. Estrogen may enhance the tubular reabsorption of calcium in the kidney in response to PTH [34,35]. Therefore, the excretion of calcium in urine in female could be lower than male even in WT mice, which was observed in the analysis of gender effect on phenotype in Ncc^R156Q/G210S mice. No difference in excretion of calcium in urine of female was observed, which could be explained by the protective effect of estrogen. As for the difference in male without significance, several possible explanations were identified. First, the standard deviation was too large in WT male mice compared with other groups. Moreover, urine collection was a crucial step. Evaporation and waste of urine occurred during urine collection. Consequently, the number of samples in the experiment must be increased, and 24 h urine must be collected in metabolic cages using a cooling device and so on. However, no difference in Ncc localization of Ncc^R156Q/G210S mice was found between male and female.

The mechanism of hypomagnesemia and hypocalciuria in GS remained incompletely understood. Reduced Mg²⁺ channel-Trpm6 abundance indicated that thiazide-induced hypomagnesemia [36] and HCTZ inhibited Ncc function in vivo. Thus, the downregulation of Trpm6 might be the mechanism of hypomagnesemia in GS. In our study, the significant decrease in Trpm6 mRNA level did not result in a decrease in protein expression in Ncc^R156Q/G210S mice, which could not provide support for the idea that reduced Trpm6 abundance might contribute to hypomagnesemia in GS. In patients with GS, reduced magnesium reabsorption caused by reduced sodium increased the excretion of magnesium. The decreased expression of Trpm6 was due to reduced sodium, but the protein level could vary because of post-transcriptional modification. Meanwhile, the function of the TRPM6 channel was dependent on proper membrane localization. TRPM6 protein levels on the apical surface of DCT cells were tightly regulated by blood EGF levels [37]. The increased expression of Trpm6 did not represent its enhanced function. On the contrary, the increased expression of Trpm6 might inhibit the transcription of mRNA. Thus, other potential mechanisms in Mg²⁺ absorption must be discovered. Epithelial calcium absorption plays a crucial physiologic role in maintaining Ca²⁺ homeostasis [38]. TRPV5 and TRPV6 were expressed at the apical membrane of Ca²⁺-transporting epithelia in the kidney, as entry channels in transepithelial Ca²⁺ transport [39]. Thus, we evaluated the expression of TRPV5 and TRPV6 in male and female. Our results indicated that the increased expression of TRPV5 and TRPV6 in male Ncc^R156Q/G210S mice compared with female Ncc^R156Q/G210S mice might account for the presence of hypocalciuria in male mice. Our research provided a new insight into the different clinical presentations in male and female patients.

We also evaluated the function of mutant Ncc in vivo. In WT mice, Ncc was located at the apical membrane of DCT cells, where Ncc reabsorbed Na⁺ and Cl⁻. As a thiazide-sensitive channel, Ncc can be inhibited by HCTZ [40]. In our functional study in vivo, mutant Ncc in Ncc^R156Q/G210S mice showed a blunted response to HCTZ confirming that mutant Ncc function was markedly diminished in compound heterozygous mice. Therefore, the significant decrease and abnormal localization of mutant Ncc in Ncc^R156Q/G210S mice accounted for their dysfunction.

Our study has certain limitations. To date, functional studies of NCC primarily focus on X.L. oocytes [4–8]. Only a few experiments have been conducted on mammalian cell lines [9]. We evaluated WT and mutant NCC surface expression and localization in MDCT cells, but we did not conduct Na⁺ uptake experiments because of the limitation of experimental conditions, although we evaluated the expression, localization, and function in vivo. Moreover, the mechanism of hypomagnesemia in our mouse model remained to be investigated.

We identified two variants from patients with GS that had not been shown to be pathogenic. We performed a series of analyses and experiments in vitro, as well as animal experiments, to confirm the pathogenicity of the two variants in the SLC12A3 gene. In addition, the novel model of Ncc^R156Q/G210S mice exhibited a typical phenotype of patients with GS except for hypocalciuria. Reduced expression and abnormal localization indicated the dysfunction of Ncc. This new model also showed gender differences in Ca²⁺ homeostasis. The increased expression of epithelial Ca²⁺ channels, TRPV5 and TRPV6, in male mice contributed to the hypocalciuria in male mice. Ncc^R156Q/G210S mice were more representative compared with Ncc knock-out mice and homozygous knock-in mice, which provided an optimal model for the further study of GS in vivo.

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