Collectrin (Tmem27) deficiency in proximal tubules causes hypertension in mice and a TMEM27 variant associates with blood pressure in males in a Latino cohort

Abstract

Collectrin (Tmem27), an angiotensin-converting enzyme 2 homologue, is a chaperone of amino acid transporters in the kidney and endothelium. Global collectrin knockout (KO) mice have hypertension, endothelial dysfunction, exaggerated salt sensitivity, and diminished renal blood flow. This phenotype is associated with altered nitric oxide and superoxide balance and increased proximal tubule (PT) Na⁺/H⁺ exchanger isoform 3 (NHE3) expression. Collectrin is located on the X chromosome where genome-wide association population studies have largely been excluded. In the present study, we generated PT-specific collectrin KO (PT KO) mice to determine the precise contribution of PT collectrin in blood pressure homeostasis. We also examined the association of human TMEM27 single-nucleotide polymorphisms with blood pressure traits in 11,926 Hispanic Community Health Study/Study of Latinos (HCHS/SOL) Hispanic/Latino participants. PT KO mice exhibited hypertension, and this was associated with increased baseline NHE3 expression and diminished lithium excretion. However, PT KO mice did not display exaggerated salt sensitivity or a reduction in renal blood flow compared with control mice. Furthermore, PT KO mice exhibited enhanced endothelium-mediated dilation, suggesting a compensatory response to systemic hypertension induced by deficiency of collectrin in the PT. In HCHS/SOL participants, we observed sex-specific single-nucleotide polymorphism associations with diastolic blood pressure. In conclusion, loss of collectrin in the PT is sufficient to induce hypertension, at least in part, through activation of NHE3. Importantly, our model supports the notion that altered renal blood flow may be a determining factor for salt sensitivity. Further studies are needed to investigate the role of the TMEM27 locus on blood pressure and salt sensitivity in humans.

NEW & NOTEWORTHY The findings of our study are significant in several ways: 1) loss of an amino acid chaperone in the proximal tubule is sufficient to cause hypertension, 2) the results in global and proximal tubule-specific collectrin knockout mice support the notion that vascular dysfunction is required for salt sensitivity or that impaired renal tubule function causes hypertension but is not sufficient to cause salt sensitivity, and 3) our study is the first to implicate a role of collectrin in human hypertension.

INTRODUCTION

Collectrin (Tmem27) is a transmembrane glycoprotein with ∼50% sequence identity with angiotensin-converting enzyme 2 (ACE2) enzyme (1, 2) but lacks any catalytic domain (2). It is highly conserved among species, sharing >80% identity between the mouse, rat, and human, and is highly expressed in the kidney in the proximal tubule (PT; 3), collecting duct (1), throughout the vascular endothelium (4), pancreatic β-cells, liver (5), intestinal epithelial cells, and retina (3, 6, 7). In the pancreas, collectrin has been shown to be cleaved by β-site amyloid precursor protein cleaving enzyme 2 (Bace2; 8, 9), which is also expressed in other tissues, including the kidney (10). The collectrin gene is located on the X chromosome, where loci encompassing collectrin in a 2-LOD interval have been linked to hypertension (HTN) and salt-sensitive HTN in the rat and human (11–14). A study in rats reported that renal collectrin expression is increased during salt-induced HTN (15). These observations raised the question of whether collectrin plays a role in HTN and salt sensitivity.

Genetic deletion of collectrin in mice revealed that it is a critical chaperone for the reabsorption of all amino acids in the PT (3, 7) as well as the uptake of l-arginine (l-Arg) in endothelial cells (4). Global collectrin knockout (KO; Tmem27^Y/−) mice display elevated blood pressure (BP) that is reduced by supplementation of l-Arg, a substrate for nitric oxide (NO) production (4). Furthermore, Tmem27^y/− mice have augmented salt-sensitive HTN that is corrected by the superoxide dismutase mimetic tempol (4). We reported that renal blood flow is significantly diminished in Tmem27^y/− mice, and renal expression of collectrin is downregulated by angiotensin II in an angiotensin type 1 receptor (AT₁R)-dependent manner (16), consistent with the observation that renal expression of collectrin is increased under high salt (15) due to suppression of renin-angiotensin II activity. Moreover, kidney cross-transplantation studies have demonstrated that loss of collectrin in the kidney augments salt-sensitive HTN (16). Loss of collectrin is also accompanied by upregulation of NHE3 in kidneys of Tmem27^y/− mice (16). These data support the notion that the upregulation of collectrin during high Na⁺ intake may be a protective mechanism to attenuate the development of salt-sensitive HTN. These observations also raise the question of whether collectrin in the endothelium or renal epithelium regulates BP homeostasis or salt sensitivity.

Here, using the Cre-loxP approach, we determined the influence of PT collectrin on arterial BP and salt sensitivity. We found that PT collectrin KO mice recapitulated the HTN but not augmented salt sensitivity seen in global Tmem27^Y/− mice. Furthermore, they displayed similar renal blood flow as wild-type (WT) control mice and enhanced endothelium-dependent vasorelaxation (EDVR), indicative of a compensatory response to systemic HTN induced by deletion of collectrin in the PT. Importantly, these data demonstrate that endothelial dysfunction is not a prerequisite for HTN, but altered renal blood flow may be a determining factor for salt sensitivity. To extend results to population studies, we examined the association of human TMEM27 single-nucleotide polymorphisms (SNPs) with BP traits in 11,926 Hispanic Community Health Study/Study of Latinos (HCHS/SOL) Hispanic/Latino participants, given their high incidence of HTN and admixture with populations that have shown salt sensitivity. We observed sex-specific SNP associations with diastolic BP (DBP).

METHODS

Animal Model

A mouse line carrying a modified Tmem27 locus with exon 4 flanked by two loxP sites was generated through the Transgenic Core facility at the University of Virginia. Two Tmem27^{tm1a(EUCOMM)Wtsi} ES cell clones (A03 and D02) were purchased from the European Mouse Mutant Cell Repository (Helmholtz Zentrum München, Neuherberg, Germany). ES cells were cultured and expanded following the protocol from the European Mouse Mutant Cell Repository. The karyotypic integrities of the clones were assessed by counting metaphase chromosome spreads. The A03 clone was found to possess 84% euploid cells, and the D02 clone was found to possess 83% euploid cells, both of which are above the threshold at which clones are deemed acceptable for injection. An individual clone of the C57BL/6N background was injected into blastocysts isolated from albino C57BL/6^{BrdCrHsd-Tyrc} mice (Envigo, Indianapolis, IN). Multiple male chimeric mice were obtained and bred with female albino wild-type mice. Germline transmission was indicated visually by the presence of black pups and confirmed by PCR genotyping. Heterozygous mice were bred with Flp mice [B6N-Tg(CAG-FLPo)1Afst/Mmucd, MMRRC 036512-UCD] to remove the FRT cassette and derive Tmem27 floxed mice. Successful germline transmission was determined by PCR (Fig. 1A). The line was then backcrossed to the 129S6 (formerly 129/SvEv) background, a salt-sensitive background, for more than 12 generations and then crossed with phosphoenolpyruvate carboxykinase (PEPCK)-Cre⁺ on the 129S6 background (kind gift from Dr. Susan Gurley) to generate a 129S6 mouse line with deletion of collectrin specifically in the PT, PEPCK-Cre⁺Tmem27^Y/Flox. Since Tmem27 is expressed throughout the S1−S3 segments of the PT (https://esbl.nhlbi.nih.gov/helixweb/Database/NephronRNAseq/All_transcripts.html), the PEPCK Cre recombinase mouse line was chosen because Cre recombinase driven by the PEPCK promoter is expressed throughout the PT (17). PEPCK-Cre⁻Tmem27^Y/Flox mice were used as controls. Male littermates of ∼3 mo of age were used for the experiments and housed in a pathogen-free facility under protocols approved by the Institutional Animal Care and Use Committee of the University of Virginia (Charlottesville, VA) and later at the University of Rochester Medical Center and in accordance with National Institutes of Health guidelines. A high-salt diet (HSD) of 6% NaCl in the form of pellets was purchased from Envigo (formerly Harlan) Teklad. Experimental models of salt sensitivity have used HSDs ranging typically between 4% and 8% Na⁺, lacking uniformity in the degree of Na⁺ loading. We have used 6% NaCl consistently in mouse models of salt sensitivity, based on our unpublished finding that a 6% NaCl diet consistently and significantly increases systolic BP (SBP) by at least 10 mmHg or 5% (an arbitrarily established criteria for salt sensitivity), as detected by tail-cuff manometry. The 6% NaCl diet was also chosen to keep experimental conditions similar to that used in global collectrin KO mice (4).

Figure 1.

Successful deletion of collectrin in the proximal tubule using phosphoenolpyruvate carboxykinase (PEPCK)-Cre. A: genotyping of floxed collectrin (Tmem27) mice showing successful germline transmission. Predicted wild-type (WT) allele: 734 bp; predicted floxed allele: 954 bp. Lanes 1 and 4, Tmem27^flox/+ (heterozygous flox female); lanes 2 and 3, WT mice; lane 5, Tmem27^flox/Y (male). B: mRNA expression of collectrin was much lower in PEPCKCre⁺Tmem27^Y/Flox mice (Cre⁺) compared with PEPCKCre⁻Tmem27^Y/Flox mice (Cre⁻ or WT). P < 0.001 by an unpaired Student’s t test. C: Western blots showing that the protein level was significantly lower in the kidney cortex in Cre⁺ mice. D: densitometry of the Western blots. E: representative immunostaining of collectrin in cortical kidney tissue showing significantly lower expression of collectrin in tubular cells in PEPCKCre⁺Tmem27^Y/Flox mice compared with PEPCKCre⁻Tmem27^Y/Flox mice. Each white arrow points to a glomerulus. ***P < 0.005.

Immunostaining

Immunostaining of collectrin was performed using paraffin-embedded kidney tissues as previously described (16). Briefly, 3- to 5-µm sections were deparaffinized and rehydrated through decreasing ethanol solutions in water. Antigen capture was performed using a commercially available unmasking solution (Vector Laboratories). Slides were incubated in blocking solution (2.4G2 in 10% horse serum, 0.1% Triton X-100, and PBS solution) and then incubated with Alexa Fluor 647-conjugated anti-mouse polyclonal collectrin antibody overnight (5 µg/mL in 10% horse serum, 0.1% Triton X-100, and PBS solution). Slides were sealed with Prolong Diamond Antifade with DAPI (Invitrogen) and visualized using a Carl Zeiss Axiovert 200M microscope with ApoTome imaging and AxioVision software (Carl Zeiss).

Immunoblot Analysis

Collectrin.

Renal cortical tissue was dissected and immediately placed into ice-cold isolation buffer (10 mM Tris, 250 mM sucrose, and 5 mM EDTA, pH 7.4) with protease inhibitor cocktail (Sigma-Aldrich). Lysates were then rapidly processed as previously described (4). Briefly, tissues or cells were homogenized using a sonicator or TissueLyser LT (Qiagen) at 50 Hz for 2 min twice, and homogenates were then spun at 12,000 rpm for 20 min at 4°C. Protein concentrations of each fraction were determined by a bicinchoninic acid (BCA) assay (Bioassay Systems). Thirty micrograms of total protein were loaded onto 12% SDS-PAGE gels and then transferred to nitrocellulose membranes per the manufacturer’s instructions (X-Cell Blot Module, Invitrogen). The membrane was blocked using Odyssey blocking buffer (LI-COR) for 1 h at room temperature. The membrane was then incubated at 4°C overnight with rabbit anti-collectrin antibody (1:1,000, custom made for Le’s laboratory by Covance Immuno Technologies, Denver, PA) as previously described (4) in blocking buffer. Secondary antibody incubation was performed for 1 h at room temperature with anti-rabbit and anti-mouse conjugated with IRDye 700 or 800 (Rockland Immunochemicals) at 1:10,000. Blots were visualized using chemiluminescent substrate (Invitrogen).

Na⁺/H⁺ exchanger isoform 3 expression.

In our previous study, which examined the expression of the major renal Na⁺ transporters and channel [Na⁺/H⁺ exchanger isoform 3 (NHE3) and NHE3-pS552, Na⁺-K⁺-2Cl⁻ cotransporter (NKCC2) and NKCC2-pT96T101, Na⁺-phosphate cotransporter 2 (NaPi2), Na⁺-Cl⁻ cotransporter (NCC) and NCC-pS71, and full-length α-epithelial Na⁺ channel (ENaC) and cleaved α-ENaC] in global collectrin KO mice, mice were fasted for 5 h to avoid dietary K⁺ affecting the expression of NCC-pS71. Although only the expression of NHE3 differed in global collectrin KO mice compared with wild-type controls, among all the transporters analyzed (16), to keep experimental conditions the same between global collectrin KO and PT collectrin KO mice, age-matched control PEPCK-Cre⁻ (n = 3) and PEPCK-Cre⁺ (n = 3) mice were fasted for 5 h (water supplied) before euthanasia as previously described (16). After the mice had been fasted, left kidneys were collected, cut in half, and snap frozen. Kidney cortices were dissected and homogenized in 1 mL of isolation buffer [5% sorbitol, 0.5 mM disodium EDTA, and 5 mM histidine-imidazole buffer, pH 7.5, with the addition of 0.2 mM PMSF, 9 µg/mL aprotinin, and 5 µL/mL phosphatase inhibitor cocktail (Sigma)]. Each sample was homogenized for 5 min at a low-speed setting with an Ultra-Turrax T25 (IKA-Labortechnik) and then centrifuged at 2,000 g for 10 min. Supernatants were retained, and the pellets were rehomogenized in another 1 mL of isolation buffer, recentrifuged, and pooled with the first supernatants. The 2,000 g supernatant (S_o) protein concentrations were determined using the BCA assay (Pierce Thermo Scientific). Samples were aliquoted and stored at −80°C.

Homogenates were denatured in SDS-PAGE sample buffer for 20 min at 60°C. Specific protein abundance and phosphorylation were assessed using primary anti-NHE3 (1:2,000) antibody [a kind gift from McDonough’s laboratory (18)] and a secondary IRDye 800 antibody (LI-COR) as described in detail elsewhere (18). Signals were detected with the Odyssey Infrared Imaging System (LI-COR) and quantified by accompanying software. Arbitrary density units were normalized to the mean intensity of the control group, defined as 1.0.

Blood Pressure Monitoring In Vivo

BP was measured in conscious mice under unrestrained conditions by radiotelemetry (TA11PA-C10, Data Sciences, St. Paul, MN) as previously described (4). Briefly, mice were anesthetized with 2–4% isoflurane and had the radiotelemetry catheter implanted into the left carotid artery. A subcutaneous pouch was made along the right flank of the animal for the caudal placement of the transmitter. Mice were housed in individual cages on receivers and allowed to recover for 7 days after implantation of the radiotelemetry device before measurements were recorded and analyzed using Dataquest A.R.T. 20 software (Data Sciences). Baseline BP was then recorded for 2 wk, followed by 2 wk of BP recording on the 6% NaCl HSD (Envigo Teklad). Reported values are expressed as means ± SD over each of the 2-wk periods.

Urine Albumin Measurements

At the end of each 2-wk period on the normal-salt diet (NSD; baseline) and HSD, mice were acclimated in individual metabolic cages for 2 days, and urine samples were collected on day 3, volume recorded, and stored at −80°C for later analysis as previously described (19). Urinary albumin was measured using the Albuwell Murine ELISA kit (Exocell, Philadelphia, PA).

Renal Hemodynamic Experiments Using Contrast Enhanced Ultrasonography

Mice were anesthetized by ketamine (90 mg/kg) and xylazine (9 mg/kg), and fur was removed by shaving and application of a depilatory. Mice were then positioned on a modified microscope stage under an ultrasound transducer held in place with a ring clamp. Prewarmed ultrasound gel was placed on the depilated skin for ultrasound application, and mouse body temperature was monitored via a rectal probe (Fine Science Tools) and maintained at 36 ± 0.5°C with a heating pad and heat lamp. A Sequoia 512 ultrasound machine with a 15L8w transducer (Acuson) was used for ultrasound experiments as previously described (16). Once the animal’s body temperature was stabilized, the right kidney was localized in real time using conventional B-mode imaging with a frequency of 14 MHz and an on-screen imaging mechanical index (MI) of 0.99. Once the kidney was localized in grayscale, contrast imaging was initiated (CADENCE function) with a frequency of 7 MHz and imaging MI of 0.16. A 60-µL bolus injection of the microbubble contrast agent (1.5 × 10⁵ microbubbles/µL) was then administered via the retroorbital plexus. Once the kidney was saturated with contrast agent and no evidence of acoustic shadowing was present, 15-s videos of microbubble destruction and replenishment were recorded. Microbubbles were destroyed using the BURST function, which consisted of a 1-s application of pulsed ultrasound with a MI of 1.9. These parameters were derived from preliminary experiments in our laboratory and were sufficient to generate a washin curve with an initial exponential return followed by a saturating plateau. After a minimum of three videos were recorded for each animal, the two-dimensional area of the kidney image was estimated by measuring the length and thickness of the kidney (using the CALIBER function). For data acquisition, the resultant images were analyzed using Sequoia analysis software (SYNGO). Tissue regions of interest were drawn, and the destruction replenishment curve was fitted to the following function: PI = A(1 − e^−βt), where PI is pixel intensity at time t, A is the maximum pixel signal during replenishment (estimate of total perfusion), and β os to the rate constant (estimate of blood velocity). β/kidney weight (in g) has been shown to correlate well with renal blood flow measurements by Doppler (20). We corrected β by the estimated two-dimensional kidney area to remove the need for euthanasia or kidney removal. The two-dimensional kidney area was calculated based on the equation for an eclipse: area (in cm²) = π × [kidney length (in cm)/2] × [kidney thickness (in cm)/2]. The estimated two-dimensional area and tissue weight after euthanasia were highly correlated (R² = 0.67). An estimate of renal blood flow was then calculated [β/kidney area (in cm²)] for each animal at baseline and again in the same animal after the HSD (paired analysis).

Endothelium-Dependent Vasorelaxation Assays

Vasoreactivity measurements were performed as previously described (4). Mice were euthanized with CO₂, and the whole mesentery was isolated and placed in Krebs-HEPES solution. The third-order mesenteric arteries were freed of the surrounding tissue and cannulated at both ends on glass micropipettes secured with a 10-0 nylon monofilament suture in a pressure myograph (Danish MyoTechnology). The third-order mesenteric arteries were maintained at 37°C in a no-flow state and held at a constant transmural pressure of 80 and 75 mmHg, respectively (21, 22). Mesenteric arteries were preconstricted with phenylephrine (PE; 10 µM), and the internal diameter was measured in response to cumulative concentrations of ACh or sodium nitroprusside (10⁻⁹−10⁻³ M). Vessel diameter was quantified after each dose of ACh using the slide book software or Danish MyoTechnology vessel acquisition software as previously described (21, 23).

Statistics

All data are presented as means ± SE. Statistical calculations were done with commercially available software packages (Minitab and NCSS). Student’s t test was used for comparisons between two groups unless otherwise stated. Differences between matched samples were analyzed by a paired t test. P values of <0.05 were considered statistically significant. P values were two sided. Two-way repeated-measures analysis was also used for SBP using SPSS (IBM). Parameter estimates are provided as means ± SE for the independent variables [“genotype” (Cre^−/− vs. Cre^+/+) and “diet” (NSD vs. HSD)].

Population Experiments

HCHS/SOL is a longitudinal study of 16,415 Hispanics/Latinos (aged 18–74 yr) recruited from households in predefined census-block groups from four United States field centers (Chicago, Miami, the Bronx, and San Diego) between 2008 and 2011 as previously described (24). Details about the complex sampling design and cohort selection for HCHS/SOL are described elsewhere (25). A baseline clinical examination included sociodemographic, clinical and behavioral assessments, and the collection of fasting blood and spot urine samples. BP was measured using an automated sphygmomanometer (OMRON HEM-907 XL, Omron Healthcare, Lake Forest, IL) on seated participants using their right resting arm following a rest period of 5 min. Cuff sizes were matched to participants using upper arm circumference measurement, and three sets of 1-min spaced measurements were taken from each person (99% completion rate). We used the average of three BP measures or all available data for the 1% of participants that did not complete all three measures (26). The study was approved by the institutional review boards at each field center and the coordinating center, and all subjects provided written informed consent. Approximately 13,000 participants consented to have DNA extracted and were genotyped using a Custom Illumina Omni2.5M array (HumanOmni2.5-8v1-1), and 11,926 participants had complete genotype and phenotype data. Quality control and estimation of principal components have been previously described (27, 28). Variants were imputed using phased haplotypes from the 1000 Genomes Project.

Statistical analyses.

We selected all SNPs within the TMEM27 gene region (55.7 Mbp within the gene, corresponding to 236 SNPs with a minor allele count of ≥30) for analyses with SBP and DBP and HTN. For individuals taking antihypertensive medications, we added 10 mmHg to measured DBP values and 15 mmHg to SBP to account for the reduction in BP due to treatment. SBP and DBP were inverse normal transformed. HTN was defined by BP > 140/90 mmHg or use of BP-lowering medications. Analyses were stratified by sex, given the differences in SNP genotypes by sex at the X chromosome (3 genotypes in women and 2 genotypes in men). We applied a generalized estimating equation approach that empirically estimates within-family correlations and complex sampling without modeling the correlation structures of complex pedigrees implemented in the SUGEN package (29). SUGEN adopts a modified version of the sandwich variance estimator, which replaces the empirical covariance matrix of the score vectors by the Fisher information matrix for unrelated subjects. Models were adjusted for age, body mass index, field center, Hispanic/Latino background [a combination of self-identified Hispanic/Latino country of origin and genetic similarity, classified as Cuban, Dominican, and Puerto Rican (Caribbean groups) and Mexican, Central American, and South American (Mainland groups)] and 10 principal components. We also compared results with sex-combined models using same covariates and adjustments for sex. The significance threshold was set to 2.1e⁻⁴ based on Bonferroni correction.

RESULTS

Successful germline transmission of Tmem27^Flox is shown in Fig. 1A. The Tmem27^Y/Flox mouse line was crossed with the PEPCK-Cre mouse line to generate PEPCK-Cre⁻Tmem27^Y/Flox (control or PEPCK-Cre⁻) mice and PEPCK-Cre⁺Tmem27^Y/Flox (PT collectrin KO or PEPCK-Cre⁺) mice. Real-time RT-PCR for mRNA expression in cortical kidney tissue and both Western blot and immunohistochemistry for protein expression demonstrated excellent efficiency of PEPCK-Cre recombinase in deleting collectrin in the PT (Fig. 1, B–E). Collectrin protein expression was reduced by nearly 90% in the kidney cortical lysate in PEPCK-Cre⁺ mice compared with PEPCK-Cre⁻ control mice. The remaining collectrin expression in PEPCK-Cre⁺ mice could be explained by the presence of collectrin in the collecting duct and endothelial cells.

Deletion of Collectrin in the PT Increases BP and Renal Superoxide Levels

By radiotelemetry, diurnal variation was intact in both control PEPCK-Cre⁻ and PEPCK-Cre⁺ mice (data not shown). SBP was significantly higher in PEPCK-Cre⁺ mice over a 2-wk period on the normal chow diet or NSD and during 2 wk on the HSD (Fig. 2A). The differences in baseline SBP on the HSD were similar to those observed between wild-type control and global collectrin knockout (TMEM27^y/−) mice (4). Although the average change in BP for each 2-wk period from the NSD to HSD was numerically higher in PEPCK-Cre⁺ mice, the difference was not statistically significant (PEPCK-Cre⁻: 15.3 ± 3.8 mmHg vs. PEPCK-Cre⁺: 19.8 ± 6.1 mmHg, P = 0.44 by Student’s t test; Fig. 2B). The difference in the average change in SBP between PEPCK Cre⁺ and Cre⁻ mice is ∼4 mmHg and is smaller than the 7 mmHg, previously observed between WT and global collectrin KO mice (4). Additional analysis using two-way repeated-measures analysis showed that PEPCK-Cre⁺ mice had greater SBP compared with PEPCK-Cre⁻ mice, with an overall average SBP of 142.0 ± 3.0 and 127.7 ± 3.0 mmHg, respectively (P = 0.001). HSD also caused a significant elevation in SBP compared with mice fed the NSD, overall (143.6 ± 2.8 vs. 126.1 ± 2.2 mmHg, P < 0.001); however, PT collectrin deficiency did not significantly influence the degree of salt sensitivity (genotype × diet, P = 0.57). There was no change in heart rate between groups or within a group on the NSD and HSD (Fig. 2C). As circadian rhythm can influence the response to an HSD (30), we also analyzed by light-dark or day (inactive) and night (active) cycles (6 AM to 6 PM and 6 PM to 6 AM, respectively). In both groups, the average daytime SBP was statistically significantly lower than nighttime, as expected. There was also a significant difference between groups in both cycles, on the NSD and HSD. However, the change in BP from the NSD to HSD during both cycles was not statistically significant between groups (see Table 1).

Figure 2.

Deletion of collectrin in the proximal tubule increases blood pressure but does not augment salt sensitivity. A: by radiotelemetry, at baseline and on a high-salt diet, the average systolic blood pressure over each 2-wk period was significantly higher in Cre⁺ compared with Cre⁻ mice [baseline (normal chow) systolic blood pressure: 120.0 ± 2.3 mmHg for Cre⁻ mice and 132.1 ± 3.2 mmHg for Cre⁺ mice (P = 0.018); high-salt diet systolic blood pressure: 135.3 ± 2.4 mmHg for Cre⁻ mice and 151.9 ± 4.5 mmHg for Cre⁺ (P = 0.013 by an unpaired Student’s t test)]. n = 6 in each group. B: change in systolic blood pressure (Cre⁻: 15.3 ± 3.7 mmHg and Cre⁺: 19.8 ± 6.5 mmHg, P = 0.57 by a Student’s t test). C: average heart rate over each 2-wk period [normal-salt diet: 592 ± 24 beats/min for Cre⁻ mice and 597 ± 27 beats/min for Cre⁺ mice (P = 0.85); high-salt diet: 570 ± 35 beats/min for Cre⁻ mice and 586 ± 33 beats/min for Cre⁺ mice (P = 0.89 by an unpaired Student’s t test)]. There was no significant change in heart rate from normal-salt diet to high-salt diet within each group by a paired t test. *P < 0.05. NS, not significant; PEPCK, phosphoenolpyruvate carboxykinase.

Table 1.

Summary of blood pressure by day and night cycles on NSD and HSD

	NSD (Baseline)		HSD		Change in Blood Pressure		P Value (Paired t Test Within Group for NSD and HSD by Cycle)
	Day Cycle	Night Cycle	Day Cycle	Night Cycle	Day Cycle	Night Cycle	Day Cycle	Night Cycle
PEPCK Cre− (n = 6)	117.2 ± 2.6	122.6 ± 2.7	132.0 ± 2.1	138.8 ± 3.3	14.9 ± 3.8	16.2 ± 4.0	0.006	0.005
PEPCK Cre+ (n = 6)	127.0 ± 3.6	137.8 ± 3.7	146.1 ± 4.8	157.8 ± 5.0	19.1 ± 6.6	20.0 ± 6.6	0.017	0.015
P value (t test between groups)	0.028	0.004	0.015	0.006	0.29	0.32

Data are presented as means ± SE for systolic blood pressure (in mmHg). HSD, high-salt diet; NSD, normal salt-diet; PEPCK, phosphoenolpyruvate carboxykinase.

As albuminuria is linked with salt sensitivity in humans and rodent models (31, 32), we assessed 24-h urine albumin excretion. At the end of each 2-wk period of NSD and HSD, and after acclimation to the metabolic cages for 2 days, we collected urine on the third day. As shown in Table 2, on the NSD, there was no difference in 24-h urine albumin excretion between groups. In both PEPCK-Cre⁻ and PEPCK-Cre⁺ mice, urine albumin excretion increased modestly and significantly from the NSD to HSD within group, but there was again no difference between groups.

Table 2.

Albumin excretion on NSD and HSD

	NSD (Baseline)	HSD	Change in Albumin Excretion	P Value Within Group
PEPCK Cre− (n = 6)	22.3 ± 4.9	70.8 ± 6.3	48.5 ± 6.2	0.0005
PEPCK Cre+ (n = 8)*	20.6 ± 4.8	58.3 ± 9.8	34.6 ± 11.7	0.008
P value between groups	0.81	0.21	0.33

Data are presented as means ± SE for urine albumin (in µg/day). Urine samples obtained for albumin excretion were from the same mice used for blood pressure measurement. *Two additional phosphoenolpyruvate carboxykinase (PEPCK)-Cre⁺ mice were added to increase power. HSD, high-salt diet; NSD, normal salt-diet.

Because Tmem27^Y/− mice have increased renal superoxide levels and decreased NO generation (4) after the HSD, we compared superoxide levels in renal tissue homogenates (by lucigenin assay) and urinary nitrate levels between PEPCK-Cre⁻ and PEPCK-Cre⁺ mice. Similar to TMEM27^y/− mice, PEPCK-Cre⁺ mice exhibited significantly higher renal superoxide levels after 2 wk on the HSD; however, their renal NO generation was similar to control mice (Fig. 3).

Figure 3.

Altered superoxide levels in proximal tubule collectrin-deficient mice. A: renal superoxide levels in the kidney cortex (measured by a lucigenin assay) after 2 wk on a high-salt diet (Cre⁻: 29.0 ± 5.1 counts/min/mg dry tissue vs. Cre⁺: 51.0 ± 6.0 counts/min/mg dry tissue, P = 0.01 by an unpaired Student’s t test). B: urinary nitrate levels (Cre⁻: 3.81 ± 0.85 µmol/day vs. Cre⁺: 4.40 ± 1.05 µmol/day, P = 0.66 by a Student’s t test). n ≥ 6 for each group. *P < 0.05. NS, not significant; PEPCK, phosphoenolpyruvate carboxykinase.

Unaltered Renal Blood Flow and Enhanced Endothelium-Dependent Vasorelaxation in PT Collectrin KO Mice

Compared with WT control mice, global TMEM27^y/− mice have diminished renal blood flow at baseline and a further reduction in renal blood flow after HSD (16). We next used the contrast-enhanced ultrasound method to determine whether the deletion of collectrin in the PT would be sufficient to alter renal blood flow. As shown in Fig. 4A, renal blood flow was similar between PEPCK-Cre⁺ and PEPCK-Cre⁻ mice at baseline and after 2 wk of HSD. It should be noted that, on the HSD, both groups had an expected decrease in renal blood flow compared with the NSD, given their salt-sensitive background (129S6). Our finding suggests that deletion of PT collectrin is sufficient to raise BP independent of altered renal blood flow and that endothelium collectrin likely contributes to renal blood flow regulation. Importantly, these data suggest that impaired renal blood flow is not a prerequisite for HTN but is required for salt sensitivity.

Figure 4.

A: renal blood flow by contrast enhanced ultrasound. Whole kidney, cortical, and medullary renal blood flow (in b/cm²) was not different between PEPCKCre⁺Tmem27^Y/Flox mice (Cre⁺) and PEPCKCre⁻Tmem27^Y/Flox mice (Cre⁻ or control) at baseline or on a high-salt diet by an unpaired Student’s t test. Within group, renal blood flow significantly dropped between normal and high-salt diets in the whole kidney and Cre⁺ mice in the cortex (*P < 0.05 by a paired t test). Whole renal blood flow: 3.3 ± 0.5 in Cre⁻ mice and 4.0 ± 0.8 in Cre⁺ mice at baseline and 2.3 ± 0.2 in Cre⁻ mice and 2.9 ± 0.8 in Cre⁺ mice on a high-salt diet. Cortical renal blood flow: 3.6 ± 0.9 in in Cre⁻ mice and 4.5 ± 0.9 in Cre⁺ mice at baseline and 2.5 ± 0.3 in Cre⁻ mice and 3.1 ± 0.9 in Cre⁺ mice on a high-salt diet. Medullary blood flow: 2.1 ± 0.5 in Cre⁻ mice and 2.8 ± 1.1 in Cre⁺ mice at baseline and 1.8 ± 0.1 in Cre⁻ mice and 2.4 ± 0.8 in Cre⁺ mice on a high-salt diet. B, left: endothelium-dependent vasorelaxation assay. Proximal tubule-specific collectrin knockout mice (Cre⁺) have enhanced vasorelaxation in response to acetylcholine (ACh). n = 5 each. *P = 0.04. B, right: endothelium-independent relaxation assay with sodium nitroprusside (SNP). No differences were observed in response to SNP between Cre⁺ and Cre⁻ mice. P = not significant. For the cumulative dose-response curves, one-way ANOVA with Sidaks multiple comparisons test was performed for all doses with *P < 0.05. PEPCK, phosphoenolpyruvate carboxykinase.

The paradigm of HTN causing vasculopathy remains a topic of debate. It has been challenged by the argument that changes in vascular function and structure may be causal in the development of HTN. Since global TMEM27^y/− mice display both HTN and endothelial dysfunction (4), we, therefore, assessed vasorelaxation capacity in PT collectrin KO mice to determine if systemic HTN would be sufficient to induce extrarenal endothelial dysfunction. Vasodilatory responses of third-order mesenteric resistance arteries in PEPCK-Cre⁺ mice were measured using pressure myography. Figure 4B, left, shows that, unlike Tmem27^Y/− mice that display impaired vasorelaxation (4), PEPCK-Cre⁺ mice displayed enhanced dilation to ACh. To determine whether vascular smooth muscle cells play a role in enhanced vasorelaxation in PEPCK-Cre⁺ mice, we performed an endothelium-independent relaxation assay using sodium nitroprusside. As shown in Fig. 4B, right, PEPCK-Cre⁺ mice and PEPCK-Cre⁻ mesenteric arteries had virtually identical vasodilatory responses to sodium nitroprusside, suggesting that PEPCK-Cre⁺ mice have enhanced endothelium-mediated dilation. This supports the notion that the enhanced vasorelaxation may be a compensatory response to systemic HTN induced by deletion of collectrin in the PT and, importantly, endothelial dysfunction is not required for the development of HTN.

Expression of Renal NHE3 Is Increased in PT Collectrin KO Mice

We have previously observed increased expression of NHE3 in TMEM27^y/− mice (16). Therefore, we performed Western blot analysis of kidney tissue lysates for NHE3 in PEPCK-Cre⁺ mice. As shown in Fig. 5A, total NHE3 protein intensity (normalized to GAPDH) was significantly higher in PEPCK-Cre⁺ mice compared with controls (PEPCK-Cre⁻ vs. PEPCK-Cre⁺: 1.00 vs. 3.14, P = 0.004). The increased NHE3 protein expression was associated with increased lithium excretion in PEPCK-Cre⁺ mice (Fig. 5B). This finding suggests increased Na⁺ reabsorption in the renal PT when collectrin is deleted in the PT and is similar to data observed in global TMEM27^y/− mice.

Figure 5.

Increased Na⁺/H⁺ exchanger isoform 3 (NHE3) expression and decreased lithium excretion in proximal tubule collectrin-deficient mice. A: by immunoblot analysis of renal cortical tissue, the abundance of total NHE3 was approximately threefold higher in Cre⁺ mice (n = 3 in each group). **P = 0.004 by an unpaired Student’s t test. B: 24-h lithium excretion under normal-salt and high-salt diets. Lithium excretion was not different between Cre⁻ (0.037 ± 0.004 mmol/L/g body wt, n = 5) and Cre⁺ (0.030 ± 0.003 mmol/L/g body wt, n = 7) mice at baseline. On the high-salt diet, lithium excretion was significantly higher than at baseline within each group by a paired t test, and was significantly lower in Cre⁺ mice (0.041 ± 0.002 mmol/L/g body wt) compared with Cre⁻ mice (0.051 ± 0.003 mmol/L/g body wt) by a Student’s t test. **P = 0.004 and *P < 0.04. BW, body weight; PEPCK, phosphoenolpyruvate carboxykinase.

Population Experiments in HCHS/SOL

The collectrin gene is located on the X chromosome, where loci encompassing collectrin in a 2-LOD interval have been linked to HTN and salt-sensitive HTN in the rat and human (11–14). We performed association analyses of SNPs within the TMEM27 gene region with BP traits (SBP, DBP, and HTN) in HCHS/SOL participants. This cohort is relatively young, with a mean age of 46-yr old (SD: 14), 41% were men, and 28% had HTN and 17% were using BP-lowering medications. Compared with women, men had high mean SBP (128 vs. 123 mmHg, men vs. women) and DBP (76 vs. 74 mmHg). Among 236 SNPs available for analyses, an intronic SNP (rs5936004) at TMEM27 was associated with unadjusted SBP and DBP in men and unadjusted DBP in women. As shown in Fig. 6, the C risk variant (minor allele) is associated with lower BPs. In analyses adjusted for age, body mass index, field center, Hispanic/Latino background, and principal components, the associations were attenuated when sex was combined (Table 1). In sex-stratified analyses, the C allele dosage was associated with lower BP, but associations were only significant in men for DBP. It should be noted that rs5936004 has a higher allele frequency in Admixed Americans in 1000 Genomes Project samples (allele frequency: 0.15) and has lower frequency in European and African ancestry (0.02–0.04). We did not identify significant associations in ACE2, which has a role in BP regulation in animal models (data not shown). This is consistent with findings from a recent gene expression analysis of >400 human kidneys that demonstrated that ACE2 was not associated with HTN (33).

Figure 6.

Systolic and diastolic blood pressures in male and female Hispanic Community Health Study/Study of Latinos participants based on rs5936004 genotypes. Analyses were performed within sex strata for males (CY, n = 625; GY, n = 4,283) and females (CC, n = 170; CG; n = 1,619; GG, n = 5,229). We applied a generalized estimating equation approach to model the association between genotypes (3 genotypes in females and 2 genotypes in males) and systolic or diastolic blood pressure. Shown are mean blood pressure values and SDs by genotypes for unadjusted models. P values for males were systolic blood pressure: 1.6e⁻⁴ and diastolic blood pressure: 3.1e⁻⁶ and for females were systolic blood pressure: 0.016 and diastolic blood pressure: 5.3e⁻⁵. Results from analyses adjusted for age, body mass index, field center, Hispanic/Latino background, and principal components are shown in Table 3.

Table 3.

236 SNPs (P < 2.1e⁴ threshold)

				Men (n = 4,908)		Women (n = 7,018)		Overall
Blood pressure trait	TMEM27 SNPs	Alleles coded/other	Coded allele frequency	β (SE)	P	β (SE)	P	P
Diastolic blood pressure	rs5936004	C/G	0.14	−0.08139 (0.02185)	1.95e−4	−0.0109 (0.0249)	0.66	1.1e−3

Data were adjusted for age, center, body mass index, and hispanic background. SNP, single-nucleotide polymorphism.

DISCUSSION

We reported that global collectrin KO mice exhibit HTN at baseline, impaired EDVR, and impaired pressure natriuresis after HSD (4). Guyton and colleagues hypothesized that the kidney’s substantial capacity for Na⁺ excretion provides a compensatory system of virtually infinite gain to oppose processes causing elevation in BP, including increases in peripheral vascular resistance. It follows that, in global collectrin KO mice, even if the initial cause of HTN is due to increased peripheral vascular resistance from reduced vasorelaxation, a defect in renal excretory function would be a prerequisite for the sustained chronic increase in BP. Because collectrin is expressed in the renal epithelia, endothelium, and brain, we assessed the relative role of renal versus extrarenal collectrin in BP regulation, using the kidney cross-transplantation approach, and reported that renal collectrin is protective against the rise in SBP and the associated renal injury after high-salt feeding (16). The defect in renal Na⁺ handling and altered pressure natriuresis observed during HSD in collectrin KO mice suggests that altered kidney function could result from a defect in renal epithelial function and/or renal hemodynamics. Indeed, we found that global collectrin KO mice display increased NHE3 expression as well as impaired renal blood flow (16).

Here, using the PEPCK Cre-lox approach, we found that deficiency of collectrin in the PT recapitulated similar levels of BP elevation as well as increased NHE3 expression as observed in global collectrin KO mice. Although higher NHE3 expression can explain the elevation of BP in PEPCK-Cre⁺ mice, the mechanism by which deletion of collectrin in the PT increases NHE3 expression is unclear but could be explained by increased oxidative stress, which has been shown to increase NHE3 expression and increase Na⁺ reabsorption in the PT (35, 36). However, whether HTN precedes oxidative stress or vice versa remains to be delineated. Compared with PEPCK-Cre⁻ control mice, PEPCK-Cre⁺ mice displayed enhanced EDVR and similar renal blood flow, suggesting that impaired PT epithelial Na⁺ handling alone is sufficient to raise BP. The enhanced EDVR in PEPCK-Cre⁺ mice is likely a peripheral vascular compensatory response to elevated BP induced by the deletion of PT collectrin. The endothelial dysfunction observed in global collectrin KO mice could be due to HTN or secondary to endothelial remodeling from collectrin deficiency (4), independent of HTN.

For decades, there has been a debate whether impaired pressure natriuresis or vasodysfunction, particularly altered renal vascular resistance, is a prerequisite for salt-induced HTN (37–39). Using the 129S6 salt-sensitive background, we found that, although there was a numeric increase in the change in BP from normal chow to HSD, PEPCK-Cre⁺ mice did not display a statistically significant augmented salt sensitivity and had virtually similarly reduced renal blood flow compared with control mice, unlike global collectrin KO mice, which exhibited augmented salt-sensitive HTN and an enhanced reduction in renal blood flow. Although it is possible that the small number of mice prevents the detection of a statistical significance, based on the means and SDs, the sample size needed to detect a significant difference, if true, would be 199/group. Furthermore, the lack of enhanced albuminuria that is linked with salt sensitivity (31, 32) is also consistent with the lack of augmented salt sensitivity in PEPCK-Cre⁺ mice. Our data support the notion that renal vasodysfunction may be a critical factor in salt-sensitive HTN but is not required for HTN. The major phenotypes of global and PT collectrin KO mice are shown in Table 4. In comparison, the lack of augmented salt sensitivity in PT collectrin KO mice is accompanied by unchanged endothelial function and renal blood flow, whereas augmented salt sensitivity observed in global collectrin KO mice is accompanied by endothelial dysfunction and decreased renal blood flow.

Table 4.

Comparison of phenotypes of mice with global and PT deletion of Tmem27 relative to their respective control mice

	Hypertension	Augmented Salt Sensitivity	Endothelial Dysfunction	Decreased Renal Blood Flow	Increased Superoxide	Increased Na+/H+ Exchanger Isoform 3 Expression	Decreased Lithium Clearance
Global collectrin knockout mice	+	+	+	+	+	+	+
PT collectrin knockout mice	+	−	−	−	+	+	+

PT, proximal tubule.

Previously, we have identified increased oxidative stress, reduced NO generation, and decreased endothelial NO synthase and neuronal NO synthase dimerization in Tmem27^Y/− mice (4, 16), which suggested a state of NO synthase uncoupling (16). Since urinary nitrates were not altered in PEPCK-Cre⁺ mice under an HSD, unlike in global KO mice in which urinary nitrates were lower than in control mice, this suggests that collectrin in the endothelium and/or collecting duct plays a role in renal NO generation that may be the determining factor in renal vascular resistance in salt-induced BP elevation. Deletion of collectrin specifically in the endothelium or collecting duct will be the subject of future studies to address this debate more definitively. It is noteworthy that increased superoxide is generally accompanied by increased urinary nitrates through increased production of peroxynitrite through reaction with NO. However, we reported that global collectrin KO mice also had increased superoxide generation yet decreased urinary nitrates on an HSD, likely due to a state of NO synthase uncoupling, where the enzyme produces superoxide instead of NO (4). In PEPCK-Cre⁺ mice, it is possible that NO synthase in the PT, primarily in the form of inducible NO synthase (40, 41), is also uncoupled and may result in a NO-deficient compartmentalized environment where superoxide, which cannot cross the cell membrane, is detoxified by superoxide dismutase or reacts with other intracellular reductive molecules, without generating peroxynitrite, and hence unchanged urinary nitrates. Importantly, although Mendelian syndromes of HTN all support distal tubular Na⁺ reabsorption as the determining factor in HTN, our finding suggests that altered PT Na⁺ reabsorption is sufficient to raise BP. Although no genetic data have supported PT regulation of BP in humans, this is supported by data on the effects on PT Na⁺ reabsorption by the now widespread use of Na⁺-glucose transporter 2 inhibitors, which have been shown to lower BP in chronic kidney disease stage 3 or 4 despite the little effect on glycosuria (42).

Genome-wide association studies for BP in humans have identified multiple loci associated with BP, but they largely excluded the X chromosome from the analysis. Therefore, associations with the TMEM27 gene with BP traits have not been assessed (https://www.ebi.ac.uk/gwas/home). To determine the effect of TMEM27 variants on BP in population studies, we chose the HCHS/SOL cohort for analysis given that the study has participants who have African Ancestry admixture in addition to European and Amerindian ancestry. In non-Hispanic Black and East Asian individuals, studies have demonstrated l-Arg-responsive salt sensitivity and HTN, respectively (43–46), suggesting an alteration in the NO pathway. Furthermore, recent research has shown that one in five participants of this relatively young United States Hispanic/Latino HCHS/SOL cohort develops HTN over a period of 6 yr of follow-up (47). The high incidence of HTN may increase the power to detect a genetic association. We identified associations with DBP in men for an SNP that is more common in Hispanic/Latino individuals, but the association was not significant with SBP, although the direction of effects was comparable to findings for DBP. Importantly, increased DBP is clinically relevant, as diastolic HTN is more prevalent in younger adults and is a major predictor of ischemic heart disease in those before the age of 60-yr old (48, 49).

Our analysis has some limitations. The intronic SNP identified in the HCHS/SOL Hispanic/Latino cohort (rs5936004) is not a functional variant, although it overlaps epigenomic annotations (H3K4me1, H3K9me3, and H3K36me3 in blood cells, the fetal kidney, and the fetal adrenal gland in ENCODE data, https://forge2.altiusinstitute.org/files/0xD619D372584211EC8C82185ABE767260/index.html). The SNP could be in linkage disequilibrium to an upstream or downstream BP effector variant that is not known. Notably, rs5936004 was not in linkage disequilibrium with SNPs in the nearby ACE2 gene. The random X chromosome inactivation and female X chromosome mosaicism could not be accounted for in female participants carrying the CG genotype (50). The number of female participants with the CC genotype was small (2.4%), which may also have limited our ability to detect significant differences. Finally, our analysis did not include salt intake or salt sensitivity. A definitive study demonstrating whether the SNP influences the expression of collectrin and an association between the expression of collectrin in the kidney and BP and salt sensitivity in humans is needed.

In summary, our study demonstrates that PT collectrin regulates BP through its role in mediating the expression of NHE3 and suggests that collectrin influences salt sensitivity by mediating the bioavailability of NO in the renal vasculature. Further studies using large samples are needed to investigate the role of the TMEM27 locus on BP and salt sensitivity in humans.

GRANTS

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant R01DK113632 (to T.H.L and N.F.).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

N.F. and T.H.L. conceived and designed research; P.-L.C., J.C.G., S.C., G.B.-K., L.C., B.E.I., and N.F. performed experiments; P.-L.C., J.C.G., S.C., Y.T.W., B.E.I., N.F., and T.H.L. analyzed data; P.-L.C., J.C.G., B.E.I., N.F., and T.H.L. interpreted results of experiments; P.-L.C., J.C.G., Y.T.W., B.E.I., N.F., and T.H.L. prepared figures; P.-L.C., J.C.G., B.E.I., N.F., and T.H.L. drafted manuscript; P.-L.C., J.C.G., J.C., B.E.I., N.F., and T.H.L. edited and revised manuscript; P.-L.C., J.C.G., S.C., G.B.-K., Y.T.W., L.C., S.W.-S., J.C., B.E.I., N.F., and T.H.L. approved final version of manuscript.