Ovarian Hormones Moderate Systolic Hypertension in Female Eln Haploinsufficient Mice

Hypertension is a hallmark of cardiovascular abnormalities associated with Williams syndrome (WS), a rare genetic disorder involving microdeletion of genes on human chromosome 7, including the elastin gene (ELN). Heterozygous deletion of Eln (Eln+/-) in mice recapitulates hypertension and arteriopathy associated with WS. Previously, differences in blood pressure elevation and sensitivity to dietary sodium were found to be less profound in female Eln+/- mice. Here, we determined whether ovarian hormones play a role in sex-related difference in blood pressure elevation resulting from Eln haploinsufficiency. Female Eln+/+ and Eln+/- mice instrumented with radiotelemetry devices were subjected to sham surgery or ovariectomy (OVX). We found that OVX lowered diastolic but not systolic blood pressure (SBP) in Eln+/- mice, resulting in increased pulse pressure. In Eln+/- mice, diuresis induced by acute volume expansion was blunted, while anti-natriuresis was exaggerated. Furthermore, amiloride lowered SBP and increased urinary Na+ excretion, suggesting that Eln+/--induced hypertension may be Na+-dependent. We conclude that increased Na+ and water retention by the kidney contribute to hypertension resulting from Eln haploinsufficiency. The underlying mechanism involves the alteration of ovarian hormone effects in the kidney and sustained signaling downstream of the V2 receptor, leading to increased ENaC activity and water reabsorption. Keywords: elastin haploinsufficiency, hypertension, ovarian hormones, renal function, vasopressin signaling. .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted November 18, 2025. ; https://doi.org/10.1101/2025.11.18.689164doi: bioRxiv preprint Graphical Abstract CDPC, cortical duct principal cell; ER, estrogen receptor; PR, progesterone receptor; ENaC, epithelial sodium channel; AQP2, aquaporin 2; V2R, vasopressin V2 receptor; ELN, elastin; PKA, protein kinase A; AC, adenylyl cyclase; Gs, stimulatory G protein; ATP, Adenosine triphosphate; cAMP, cyclic AMP; Å, activating; , inhibiting .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted November 18, 2025. ; https://doi.org/10.1101/2025.11.18.689164doi: bioRxiv preprint

Hypertension is a leading risk factor for the development of cardiovascular disorders and chronic kidney disease and is notably more prevalent in post-menopausal women but less in pre-menopausal women compared to age-matched males1,2. Several studies establish the anti-hypertensive effects of ovarian hormones as a primary biological difference between males and females3,4. Ovarian hormones, particularly estrogen, modulate cardiovascular function by activating endothelial nitric oxide synthase (eNOS) and influencing the activity of the renin-angiotensin system (RAS)5-8. Furthermore, estrogen receptors localized on renal epithelial cells regulate the activity and expression of sodium channels and transporters along the nephron9-11, suggesting a role for sex hormones in the regulation of fluid and electrolyte balance, which serves as a primary mechanism by which the kidney maintains long-term blood pressure control. However, the precise mechanisms by which sex hormones confer such protective effects, either by acting in the kidney or in other organ systems, are incompletely understood. Long-term blood pressure control by the kidney is a complex physiological process involving the regulation of renal hemodynamics and controlled tubular reabsorption to maintain extracellular fluid volume and electrolyte balance12,13. Central to this process is renal autoregulation, which is mediated by kidney intrinsic mechanisms that are in turn finetuned by factors that are either intrinsic or extrinsic to the kidney. Renal autoregulation is key to adjusting renal vascular resistance (RVR) to stably maintain renal blood flow (RBF) and glomerular filtration rate (GFR), despite fluctuating changes in systemic blood pressure14,15. Abnormal autoregulation can potentially lead to renal ischemia or barotraumatic damage of the renal microvasculature and the glomerular capillary network14,16. Such damage and autoregulation failure could lead to hyperfiltration and over-saturation of the renal tubular system, thereby causing proteinuria and/or inappropriate loss or excessive retention of electrolytes and water sustaining hypertension15,17. Chronic hypertension elevates renal perfusion pressure and is associated with elevated peripheral vascular resistance and stiffening, that together are implicated in the impairment of renal autoregulation. Increased vascular stiffness develops partly from the loss of functional elastin (ELN), the principal extracellular matrix (ECM) protein in vascular elastic fibers that form the internal, medial, and external elastic laminae18,19. ELN is produced and secreted into the ECM as tropoelastin monomers, which concatenate in structured fibrillar networks of other ECM proteins and are finally crosslinked by lysyl oxidase to form mature elastin fibers20,21. Mature ELN is formed in early life but fatigues with age and is also gradually fragmented and degraded by secreted elastases and local matrix metalloproteinases (MMPs), thus progressively losing its initial mechanical properties without a replacement22-24. ELN degradation also increases collagen .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted November 18, 2025. ; https://doi.org/10.1101/2025.11.18.689164doi: bioRxiv preprint (COL) deposition, thereby increasing COL/ELN ratio22,23,25. As such, disease- and age-related vascular remodeling, including arterial stiffening, calcification, and dysfunction are also associated with deficiency of functional ELN and tissue fibrosis26-28. Previously, we reported that Eln haploinsufficiency (Eln+/-) in mice is independently associated with increased RVR and pathological remodeling of the renal microvsculature29. These structural and functional changes were shown to precede the onset of hypertension that becomes fully established in Eln+/- mice by 1 month of age30. Furthermore, the hypertensive phenotype in Eln+/- mice was found to be more pronounced in male than female mice, when compared to their respective Eln+/+ controls, suggesting a potential role for sex-related differences, including differences in sex hormone levels, in mitigating or driving hypertension in Eln haploinsufficiency. However, the relationship between functional ELN level and the biological effects of sex hormones in the pathogenesis of hypertension has not been thoroughly explored. Previously, we showed that accelerated structural changes in the renal microvasculature associated with Eln haploinsufficiency exacerbate age-related impairment of renal hemodynamics in female mice31. Whether Eln haploinsufficiency leads to the disruption of the homeostatic regulation of water and electrolyte balance by the kidney to contribute to chronic blood pressure elevation has not been fully explored. Furthermore, whether changes in sex hormone levels impinge on the effects of Eln haploinsufficiency to drive sustained blood pressure elevation is also unknown. In this study, we postulated that ovarian hormones mitigate blood pressure elevation in Eln+/- female mice by fine-tuning renal tubular handling of water and sodium. To test this hypothesis, we investigated the effects of ovarian hormone depletion, via ovariectomy (OVX) in wild type (Eln+/+) and Eln+/- female mice, on diurnal blood pressure and fluid and sodium handling by the kidney. Our results show that Eln haploinsufficiency has differential effects on the response of systolic and diastolic blood pressure to ovarian hormone depletion, which might be related to effects on renal tubular sodium and water handling. .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted November 18, 2025. ; https://doi.org/10.1101/2025.11.18.689164doi: bioRxiv preprint

Animals The study was performed under a protocol (# 2020-0025) approved by the Institutional Animal Care and Use Committee of Case Western Reserve University. Experiments were performed using 3–4-month-old female wild-type (Eln+/+) and elastin heterozygous (Eln+/-) mice that have been backcrossed several generations into the C57 Bl/6 genetic background (Charles River, MA). The generation of Eln+/- mice has been described previously30,32. Mice were provided access to food and water ad libitum in our institution’s animal resource center at 22°C with a 12-h light/dark cycle. Ovariectomy and sham procedures All appropriate steps were taken to perform aseptic surgery for oophorectomy/ovariectomy (OVX) as described previously, with slight modifications (supplemental Figure 1A)33-35. Briefly, mice were shaved on the dorsal midline and an incision of ~1 cm was made. The skin was detached from the adjoining muscle wall using blunt dissection, followed by a 10-mm incision through the abdominal wall. The ovary was externalized and removed by cauterization between the end of the uterine horn, the ovary and ovarian artery. The abdominal wall was closed with an absorbable suture and the skin closed with metal staples. The same procedure was repeated on the other side to remove the second ovary, and finally, animals were injected with meloxicam as part of post-operative pain management. Sham-operated animals were subjected to the same surgical procedure, except that the ovaries were exteriorized briefly but not excised. The success of the ovariectomy was confirmed by uterine atrophy, determined by harvesting and weighing the uterus post-euthanasia and normalizing to body weight. Ovariectomized mice with marginal uterine atrophy relative to sham controls were excluded from the study (supplemental Figure 1A – 1C). The mice were used for experiments at least 4 weeks post-ovariectomy or sham surgery. Diurnal blood pressure and heart rate monitoring using radiotelemetry Conscious blood pressure data were acquired using radiotelemetry (HD-X10, Data Sciences International, St. Paul, MN), as described previously29,36. Briefly, the pressure-sensing catheter of the telemeter was implanted in the left carotid artery and advanced into the aortic arch, while the transmitter body was placed subcutaneously, under isoflurane anesthesia. After a 7-day recovery period, baseline blood pressure and heart rate were recorded for 24 hours. Subsequently, mice underwent ovariectomy, and blood pressure, heart rate, and locomotor activity were recorded at least 4 weeks after ovariectomy. .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted November 18, 2025. ; https://doi.org/10.1101/2025.11.18.689164doi: bioRxiv preprint Dietary sodium modification and diurnal blood pressure and heart rate recording Sham-operated and OVX mice were placed on standard mouse chow (0.22% sodium; 5P76/P3000, LabDiet, Richmond, IN) or on a sodium-deficient diet (0.01% sodium; Invigo, cat. #: TD.90228) for 10 days, after which blood pressure and heart rate were recorded for 24 hours. The mice then received amiloride (1.5 mg/kg. i.p.) once daily for 3 days followed by 24-hour radiotelemetry recording and overnight urine collection using a mouse metabolic cage. Glomerular filtration rate measurement in conscious mice Glomerular filtration rate (GFR) was assessed using the transdermal method, as previously described, with minor modifications37. Mice were initially implanted with a polyethylene-10 (PE-10) catheter into the jugular vein for fluorescein-isothiocyanate (FITC)-labeled sinistrin administration. The hair at the nape was shaved and disinfected, followed by small midline incisions made at the ventral and dorsal regions of the neck. A metal trocar was cautiously utilized to create a tunnel from the front to the back of the neck, through which the PE-10 catheter was threaded. The subcutaneous tissue was carefully dissected to expose the jugular vein, where the PE-10 catheter was inserted and secured with a 5-0 silk. The catheter was flushed with a sterile 10% heparinized saline solution to confirm proper placement and to prevent blood clot formation. The section of the catheter extending to the nape was sealed using a cauterizer and then secured with suture and tissue glue. To prepare the mouse for GFR measurement, the flank of the animal was shaved, followed by the application of hair removal cream before attaching the GFR mini device to an adhesive patch. The device was fastened with tape and allowed to record a steady baseline reading for ~5 minutes before administering a bolus of FITC-sinistrin (35 mg/mL in saline) via the jugular vein catheter. Transcutaneous fluorescence was recorded for 2 hours, and the acquired data was used to calculate GFR using the manufacturer-provided software (MB_Lab2, Medibeacon). GFR values were normalized to body weight. Renal blood flow procedure Simultaneous renal blood flow (RBF) and blood pressure were measured under isoflurane anesthesia in sham and OVX Eln+/+ and Eln+/- mice using a well-established technique, as previously described38. Briefly, catheters were surgically implanted into the left carotid artery and right jugular vein under aseptic conditions. Following this procedure, a left flank incision was made to expose the left renal artery. A perivascular flow probe (0.5PSB Nanoprobe with handle, Transonic, Ithaca, NY) was carefully positioned around the left renal artery to enable real-time measurement of RBF. Baseline blood pressure and RBF were continuously recorded using .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted November 18, 2025. ; https://doi.org/10.1101/2025.11.18.689164doi: bioRxiv preprint PowerLab 8/35 (ADInstruments, CO) at a sampling rate of 1000 Hz for at least 20 minutes. Overnight urine collection Mice were individually housed in metabolic cages overnight for urine collection, with free access to food and water. Urine volume was determined gravimetrically. Osmolality was measured using an osmometer (Wescor Vapor Pressure Osmometer VAPRO, model 5520), and sodium and potassium levels were assessed by flame photometry. Urine flow rate, and sodium and potassium excretion rates were normalized to body weight in grams. Acute volume loading Under light isoflurane anesthesia, mice received subcutaneous injection of normal saline solution pre-warmed to 37°C, equivalent to 6% of their body weight. Urine excretion rate was assessed by placing the volume-loaded mice in metabolic cages, and urine samples were collected at 75- and 150-minutes post saline administration. This procedure was conducted both in the presence and absence of the selective vasopressin type 2 receptor (V2R) antagonist, tolvaptan (Tocris, Minneapolis, MN), administered at a dose of 1 mg/kg via intraperitoneal injection shortly before the subcutaneous saline loading. Plasma analysis Mice were anesthetized and administered heparin before collecting blood from the left ventricle of the heart. After collection, blood was centrifuged at 4.5xg for 5 minutes at 4°C to separate plasma. Plasma osmolality was measured using an osmometer, while plasma copeptin was measured using a mouse copeptin ELISA kit by following the manufacturer’s instructions (LS BIO, Shirley, MA). Statistical Analysis Data are presented as mean ± standard error of the mean (SEM). Sidak post hoc test was used following a two-way or three-way ANOVA for multiple comparisons between the experimental and control groups, where appropriate. All statistical analyses were performed using GraphPad Prism version 9 (GraphPad Software, San Diego, CA). .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted November 18, 2025. ; https://doi.org/10.1101/2025.11.18.689164doi: bioRxiv preprint

Ovariectomy exacerbates blood pressure elevation in Eln+/- female mice Previously, we showed that Eln+/- mice of both sexes exhibit higher nighttime systolic blood pressure (SBP) compared to their Eln+/+ counterparts29. We also reported that blood pressure elevation was relatively more pronounced in male Eln+/- mice than in female Eln+/-, when compared to their respective controls, suggesting sexual dimorphism in the blood pressure phenotype resulting from Eln haploinsufficiency29. To test whether ovarian hormones play a role in the phenotypic differences, we examined diurnal blood pressure by radiotelemetry before and after OVX in Eln+/+ and Eln+/- mice. Before OVX, Eln+/- female mice exhibited higher nighttime SBP and slightly elevated diastolic blood pressure (DBP) compared to Eln+/+ mice, with no discernible difference in heart rate between the groups (Figure 1). Four weeks following OVX, nighttime SBP was slightly elevated in both genotypes, while daytime SBP was further elevated by ~10 mmHg only in Eln+/- mice (Figure 1A and supplemental Figure 2A). In contrast, nighttime DBP showed a downward trend after OVX in Eln+/- mice, whereas daytime DBP was relatively unchanged in both genotypes (Figure 1B and supplemental Figure 2B). The augmented SBP elevation in OVX Eln+/- mice was confirmed to be independent of changes in locomotor activity, as both genotypes showed similar patterns in diurnal locomotor activity, before and after OVX (supplemental Figure 2C). Heart rate was similar between the two groups, with equal magnitude of decline following OVX (Figure 1C), while pulse pressure, an indicator of arterial stiffness, was elevated in both genotypes but appeared to be relatively more pronounced in Eln+/- mice (Figure 1D). Together, these results indicate that ovarian hormones moderate systolic hypertension associated with Eln haploinsufficiency in female mice. .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted November 18, 2025. ; https://doi.org/10.1101/2025.11.18.689164doi: bioRxiv preprint Eln haploinsufficient mice are insensitive to the anti-natriuretic effect of ovariectomy Ovarian hormones, including estrogen and progesterone, have been shown to affect renal vascular tone and renal epithelial sodium transport through the activation of surface receptors and by their genomic effects39-43. The vascular and epithelial effects of ovarian hormones can alter renal perfusion and tubular function44-46. Therefore, we examined the effects of OVX on RBF and pressure-natriuresis relationship to determine whether Eln haploinsufficiency alters the effects of ovarian hormone depletion by OVX on renal hemodynamics and tubular function to exacerbate systolic hypertension. At baseline, sodium excretion rate trended high in sham Eln+/- and OVX Eln+/+ mice, compared sham Eln+/+ mice (Table 1, P = 0.098 and 0.063, respectively). However, both plasma and urine osmolality were similar between all groups. In sham mice, RBF trended Figure 1. Diurnal blood pressure and heart rate of Eln+/+ and Eln+/- mice before and after ovariectomy (OVX). (A – C) 24-hour systolic and diastolic blood pressure, and heart rate of Eln+/+ and Eln+/- female mice before and 4 weeks after OVX. (C) A decrease in heart rate was observed in both groups after OVX. (D) Pulse pressure, an indicator of arterial stiffness, increased in both groups after OVX. Data are presented as mean ± SEM; Baseline Eln+/+ (n=10), Baseline Eln+/- (n=13), OVX Eln+/+ (n=8), OVX Eln+/- (n=8). *P<0.05, between-group comparison, analyzed using a two-way ANOVA mixed model with Sîdak post hoc multiple comparison tests. Sham Ovariectomy Genotype Eln+/+ Eln+/- Eln+/+ Eln+/- Number of animals 6 7 6 9 Urine flow rate (ul/hr/g BW) 1.04 ± 0.20 1.35 ± 0.32 1.46 ± 0.33 1.70 ± 0.52 Urinary Na+ excretion rate (mmol/hr/g BW) 182 ± 42 362 ± 85 386 ± 52 295 ± 74 Urinary K+ excretion rate (mmol/hr/g BW) 695 ± 200 867 ± 240 807 ± 137 750 ± 235 Urine osmolality (mmol/Kg) 2833 ± 410 2263 ± 231 2709 ± 365 2151 ± 342 Plasma osmolality (mmol/Kg) 335 ± 20 340 ± 4 374 ± 27 374 ± 11 Table 1: Comparative analysis of urine flow rate, urinary Na+ and K+ excretion rates, and urine and plasma osmolality in sham and ovariectomized Eln+/+ and Eln+/- mice. LKW, left kidney weight. Values are mean ± SEM .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted November 18, 2025. ; https://doi.org/10.1101/2025.11.18.689164doi: bioRxiv preprint slightly higher in Eln+/- relative to Eln+/+ mice; However, RBF was substantially elevated in Eln+/- compared to Eln+/+ mice, following OVX (Figure 2A). Renal vascular resistance (RVR) was similar among the different groups but showed a decreasing trend in Eln+/- mice after OVX (Figure 2B). GFR was similar between the two genotypes and trended downwards only in Eln+/+ mice after OVX, despite the augmented RBF in Eln+/- mice after OVX (Figure 2C). In sham mice, filtration fraction (FF) trended lower in Eln+/- mice compared to Eln+/- mice and, following OVX, decreased further in both genotypes (Figure 2D). Furthermore, following OVX, the resulting daytime (Figure 2E) and nighttime (Figure 2F) pressure-natriuresis relationship showed a more rightward shift with little change in the slope in Eln+/- mice compared to Eln+/+ mice. These trends were found to be consistent with increased sodium retention and salt-sensitive hypertension13,47-51. Figure 2. Renal hemodynamics and pressure-natriuresis in sham and ovariectomized (OVX) Eln+/+ and Eln+/- mice. (A) Renal blood flow (RBF) was significantly increased in OVX Eln+/- mice, while (B) renal vascular resistance (RVR) was similar among the different groups but trended lower Eln+/- mice after OVX. (C) GFR trended low in Eln+/+ OVX mice but was similar among the other groups. (D) Filtration fraction (FF), calculated as the ratio of GFR to RPF, trended low in sham Eln+/- mice and following OVX in both Eln+/+ and Eln+/- mice. Pressure-natriuresis curves generated with daytime (E) and nighttime (F) systolic blood pressure (SBP) and overnight urine sodium excretion rates of pre- and post-OVX in Eln+/+ (n= 4-8) and Eln+/− (n= 5–6) mice. Data are presented as mean ± SEM. UvNa+, urinary sodium excretion rate. LKW - Left kidney weight. **P<0.01 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted November 18, 2025. ; https://doi.org/10.1101/2025.11.18.689164doi: bioRxiv preprint Elevated systolic blood pressure in Eln+/- mice is insensitive to low-salt diet As previously reported, blood pressure elevation in Eln+/- female mice is modest and insensitive to high-salt diet29. However, based on the observation herein that OVX enhanced SBP elevation in Eln+/- mice, and that overnight natriuresis in female Eln+/- mice phenocopied Eln+/+ after OVX (Table 1), we postulated that blood pressure elevation resulting from Eln haploinsufficiency in female mice is due, at least partially, to abnormal sodium and water handling by the kidney. To test this hypothesis, we examined the effect of low-salt diet (LSD) on SBP in Eln+/- female mice by placing sham and OVX mice on LSD for 10 days and reassessed diurnal blood pressure. As shown in Figure 3A, there was a slight downward trend in daytime SBP in sham Eln+/+ mice on LSD but no change in sham Eln+/- cohort. In Eln+/- OVX mice fed LSD, SBP remained elevated relative to their Eln+/+ cohort (Figure 3B). Conversely, DBP was similar in both sham and OVX groups of both genotypes on LSD (Figures 3C & 3D). Baseline heart rate was similar between the two genotypes, with or without OVX, and appeared to decrease at similar rates after LSD (supplemental Figure 3). Low-salt diet-induced augmentation of RAS activity is enhanced by OVX in Eln+/- mice Figure 3. Effect of low-salt diet on diurnal blood pressure in sham and OVX Eln+/+ and Eln+/- mice. Low-salt diet slightly lowered SBP in Eln+/+, thereby enhancing the difference in SBP between (A) sham and (B) OVX mice relative to their respective cohorts. Diastolic blood pressure in (C) sham and (D) OVX mice was similar. Data are presented as mean ± SEM, *P<0.05, between-group comparison, analyzed using a two-way ANOVA mixed model with Sîdak post hoc multiple comparison tests. .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted November 18, 2025. ; https://doi.org/10.1101/2025.11.18.689164doi: bioRxiv preprint Previous studies showed that RAS activity is augmented in Eln+/- mice and contributes to the hypertensive phenotype at least partly by influencing vascular tone regulation29,30. However, whether augmented RAS activity by itself, or combined with the effects of ovarian hormones, enhances sodium reabsorption by the kidney, thereby contributing to sustained blood pressure elevation in Eln+/- mice was unclear. Because LSD failed to lower SBP in OVX Eln+/- female mice, and because LSD has been shown to induce a compensatory increase in renal sodium reabsorption via RAS activation52, we tested whether blood pressure insensitivity to changes in dietary sodium was due to maladaptive increase in RAS activity. We evaluated RAS activity by assessing blood pressure response to acute systemic blockade of angiotensin-converting enzyme (ACE) with captopril in conscious sham and OVX mice on LSD. In sham animals, SBP and DBP in Eln+/- mice decreased by ~30 mmHg within 30 minutes of captopril administration and remained low for more than 1 h post injection, whereas Eln+/+ mice exhibited a maximum blood pressure decline of ~15 mmHg within 60 minutes of captopril injection (Figure 4A). The depressor response to captopril was accompanied by tachycardia in sham Eln+/- but not Eln+/+ mice (Figure 4E). In OVX mice, LSD appeared to elicit a more robust and rapid depressor response to captopril injection in Eln+/- (~40-mmHg decline in SBP) relative to Eln+/+ mice (Figure 4B & 4D). As in sham animals, the depressor response to captopril was accompanied by tachycardia, which was less robust in Eln+/- relative to Eln+/+ mice (Figure 4F). Overall, our data suggest that ovarian hormone depletion increases the sensitivity of the RAS to LSD and is further enhanced by Eln haploinsufficiency. Sustained elevation of SBP in OVX Eln+/- mice on LSD is associated with marked suppression of natriuresis and diuresis To determine the basis of SBP elevation and blood pressure insensitivity to LSD in Eln+/- mice, we assessed urine flow rate and urinary sodium and potassium excretion rates in female mice on normal chow or LSD after sham and OVX. In sham Eln+/- mice on normal salt diet (NSD), overnight urine flow rate was higher, while urine osmolality was lower compared to the respective sham Eln+/+ mice (Supplemental Figure 4A). Following LSD, urine flow rate increased markedly in the Eln+/+ mice, whereas it exhibited a decreasing trend Eln+/- mice (Supplemental Figure 4A). Increased urine flow rate in Eln+/+ mice was accompanied by a decreasing trend in urine osmolality, whereas a lower urine flow rate in sham Eln+/- mice was accompanied by a slightly increased trend in urine osmolality (Supplemental Figure 4C). In OVX mice, both urine flow rate and urine osmolality under NSD were similar between Eln+/+ and Eln+/- mice. However, after LSD, urine flow rate showed a decreasing trend at similar rates in both genotypes, while urine osmolality .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted November 18, 2025. ; https://doi.org/10.1101/2025.11.18.689164doi: bioRxiv preprint appeared to trend differently (Supplemental Figure 4B and 4D). When compared to their sham counterparts on NSD, OVX increased urine flow rate with very little effect on urine osmolality in Eln+/+ OVX mice, and the effect of OVX on urine flow rate was slightly lowered by LSD (Supplemental Figure 4A vs 4B and 4C vs 4D). Conversely, both OVX and LSD did not appear to alter urine flow rate and urine osmolality under normal chow or LSD conditions in Eln+/- mice (Supplemental Figure 4). On NSD and prior to OVX, overnight sodium excretion rate in sham Eln+/- mice showed an upward trend relative to values in sham Eln+/+ mice. However, following OVX, sodium excretion rate Figure 4. Blood pressure and heart rate response to acute systemic captopril administration in conscious mice instrumented with radiotelemetry devices. Baseline measurements were collected for ~1 hour prior to captopril administration (10 mg/kg, i.p.). Average values over 30 minutes preceding the injection were subtracted from pre- and post-injection blood pressure and heart rate values. Δ Systolic blood pressure in sham (A) and OVX mice (B). Δ Diastolic blood pressure in sham (C) and OVX (D) mice. Δ Heart rate in sham (E) and OVX (F) mice. Data are presented as mean ± SEM. The time of injection is denoted by red arrow. ShamOVX0 200 400 600 800UNa+ (µmol/ml/h/g BW) WT_NSDWT_LSD ns ✱✱ Eln+/- _NSDEln+/- _LSD ✱✱✱ns ShamOVX05001000150020002500UK+ (umol/ml/g BW) WT_NSDWT_LSD ns ns Eln+/- _NSDEln+/- _LSD ns ns ShamOVX200 300 400 500 600Plasma Osmolality (mmol/Kg) WT_NSDWT_LSD ns ns Eln+/- _NSDEln+/- _LSD ns ns A B C .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted November 18, 2025. ; https://doi.org/10.1101/2025.11.18.689164doi: bioRxiv preprint increased only in Eln+/+ relative to Eln+/- mice on NSD (Figure 5A). Conversely, LSD led to a 4-fold decrease in sodium excretion rate in sham Eln+/+ mice relative to ~16-fold decrease in sodium excretion rate in sham Eln+/- mice when compared to their respective NSD controls (Figure 5A). LSD equally lowered sodium excretion rate in Eln+/+ OVX mice compared to sham Eln+/+ control, whereas it slightly enhanced overnight sodium excretion rate in Eln+/- OVX relative to Eln+/- sham mice and to a similar level in Eln+/+ OVX mice (Figure 5A). Eln insufficiency or OVX had no effect on urinary potassium excretion rate in mice on NSD; However, LSD tended to lower potassium excretion rate in Eln+/+ OVX mice and both groups of Eln+/- mice (Figure 5B). Plasma osmolality trended low after LSD in sham and OVX mice of both genotypes but were not significantly different from mice on NSD. Overall, these results suggest that i) LSD unmasks increased sodium reabsorption by the kidney due to Eln haploinsufficiency, and that ii) depletion of ovarian hormones minimizes the differences between Eln+/+ and Eln+/- mice regarding renal sodium and water handling, implying that ovarian hormones mitigate renal tubular dysfunction resulting from Eln haploinsufficiency in female mice. Amiloride reduces daytime SBP and promotes natriuresis in Eln+/- mice fed low salt diet Elevated RAS activity increases blood pressure in part by promoting ENaC synthesis and insertion in principal cells of the distal nephron, thereby increasing sodium and water reabsorption53. As shown above, both OVX and Eln haploinsufficiency enhanced RAS activity in female mice. Therefore, to test whether sodium reabsorption via ENaC is involved in the exacerbation of hypertension upon ovarian hormone depletion in Eln+/- female mice, we examined the effect of subchronic administration of the ENaC inhibitor, amiloride, on diurnal blood pressure in OVX mice fed low salt diet. As shown in Figure 6, subchronic amiloride administration reduced SBP and DBP in both Eln+/+ and Eln+/- OVX mice. The effect of amiloride on blood pressure was more evident in daytime SBP of Eln+/- OVX mice, thereby narrowing the difference between the two genotypes in this period. Overnight urine sodium excretion rate trended higher in Eln+/- OVX mice following amiloride administration (Figure 6G). However, amiloride had no effect on urine osmolality, though it lowered urine potassium excretion rate equally in both genotypes after OVX Figure 5. Low sodium diet exaggerates Na+ reabsorption in Eln+/- female mice. (A & B) The effect of low salt diet on overnight urine Na+ and K+ excretion rates in sham and ovariectomized (OVX) mice on standard chow (NSD) or low sodium diet (LSD). (C) The effect of LSD on osmolality of plasma obtained from sham and OVX mice at the end of 10 days on NSD or LSD. Data in bar graphs are expressed as mean ± SEM. Symbols represent individual animals in each experimental group and genotype. Between-group comparisons were analyzed using 3-way ANOVA mixed model with Sîdak post hoc multiple comparison tests. **, ***P<0.01, 0.001; ns, not significant. .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted November 18, 2025. ; https://doi.org/10.1101/2025.11.18.689164doi: bioRxiv preprint (Figure 6H). These results suggest that increased sodium reabsorption via ENaC contributes to the exacerbation of blood pressure elevation in OVX Eln+/- female mice. .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted November 18, 2025. ; https://doi.org/10.1101/2025.11.18.689164doi: bioRxiv preprint Eln haploinsufficiency suppresses excess sodium excretion in response to vasopressin receptor blockade after acute fluid volume expansion The vasopressin system is widely recognized as the primary determinant of urine osmolality due to its regulation of water reabsorption in the collecting duct54. However, vasopressin can also affect sodium reabsorption by upregulating ENaC activity in the principal cells of the connecting tubule and collecting duct55-57. Indeed, certain mutations in vasopressin V2 receptor (V2R) that lead to increased ENaC activity are implicated in volume-dependent hypertension56,58. Therefore, we determined whether Eln haploinsufficiency promotes sodium retention by altering the effects of vasopressin on renal sodium and water handling. We examined urine flow rate and urinary sodium excretion rate following acute extracellular fluid volume expansion by a bolus administration of normal saline (6% of body weight, SQ), in the presence or absence of the V2R antagonist, tolvaptan (1 mg/kg, i.p.). In saline-treated sham and OVX Eln+/+ and Eln+/- mice, urine flow rate was similar 75 minutes after acute volume loading (Figure 7A and 7B). Interestingly, fewer Eln+/- mice produced urine at 75 minutes compared to their respective Eln+/+ controls 0751500.00.10.20.30.40.50.60.70.8 Time (mins) Urine Flow Rate (µL/min/g BW) Sham Eln+/+ (5) SalineSham Eln+/- (6) SalineSham Eln+/+ (5) TolvaptanSham Eln+/- (6) Tolvaptan ✱✱ # 0751500.00.10.20.30.40.50.60.70.8 Time (mins) Urine Flow Rate (µL/min/g BW) OVX Eln+/+ (6) Saline OVX Eln+/- (9) SalineOVX Eln+/+ (6) TolvaptanOVX Eln+/- (9) Tolvaptan ✱✱ # SalineTolvaptanSalineTolvaptanSalineTolvaptanSalineTolvaptan 0 50 100 150UNa+ (µmol/ml/min/g BW) UNa+ after Volume Loading_Sham & OVXWT_Sham WT_OVX <0.0001 0.0004Eln+/- _Sham<0.0001 Eln+/- _OVX 0.9641 A B C Figure 6. ENaC blockade with amiloride decreases SBP in OVX Eln+/- mice fed low salt diet. Amiloride was administered daily (1.5 mg/kg, i.p.) for 3 days at least 4 weeks after ovariectomy (OVX) and at the end of 10 days of low salt diet in wild type (Eln+/+, n=5) and Eln+/- (n=6) mice. Twenty-hour systolic (SBP, A & B) and diastolic (DBP, C & D) blood pressure was measured one day before and on the last day of amiloride injection. Overnight urine flow rate (E), osmolality (F), sodium excretion rate (G), and potassium excretion rate (H) before and after 3-day administration of amiloride in Eln+/+ and Eln+/- OVX mice. Data points in bar graphs represent individual animals in each experimental group and genotype. Values in line graphs and bar graphs are expressed as mean ± SEM. **P<0.01, between-group comparison, analyzed using two-way ANOVA mixed model with Sîdak post hoc multiple comparison tests. NS, not significant. Figure 7. Urine flow and sodium excretion rate following acute extracellular fluid volume expansion with normal saline in the presence and absence of tolvaptan (1 mg/kg, i.p.). Urine flow rate after volume loading with saline, both with and without tolvaptan, are presented for sham-operated (A) and OVX (B) mice. (C) bar graph showing sodium excretion rate, which was calculated using the concentration of sodium in the urine collected from the 150th minute. Circles and solid lines represent urine flow rates without tolvaptan, while squares and dashed lines indicate rates with tolvaptan. Data are presented as mean ± SEM. **P<0.01, vs values at the 150th minute urine flow rates between Eln+/+ and Eln+/- mice treated with tolvaptan. #P<0.05, urine flow rates between the 75th and 150th minute time points in mice given tolvaptan. .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted November 18, 2025. ; https://doi.org/10.1101/2025.11.18.689164doi: bioRxiv preprint (supplemental Figure 5). In the presence of tolvaptan, urine flow rate in sham and OVX Eln+/+ mice increased substantially at 75 minutes and was further elevated at 150 minutes. In contrast, urine flow rate in Eln+/- mice was slightly elevated by tolvaptan only at 75 minutes in sham but not OVX animals (Figure 7A vs. 7B). In sham Eln+/+ mice, tolvaptan increased urinary sodium excretion after acute volume loading, and this effect was abolished in Eln+/+ OVX mice (Figure 7C). By contrast and similar to urine flow rate, tolvaptan had no effect on urinary sodium excretion rate in Eln+/- mice, after either sham or OVX (Figure 7C). These results indicate that Eln haploinsufficiency alters the sensitivity of the renal vasopressin system to acute sodium and water overload. The results also suggest that Eln haploinsufficiency enhances vasopressin signaling-mediated sodium, and thus water, reabsorption by the kidney. Discussion Elastin is expressed predominantly in the ECM of extensible organs such as the skin, lung, and conduit arteries, where it confers compliance21,59. In the kidney vasculature, ELN expression is confined to elastic fibers in small, multilayered preglomerular arteries60. Hemizygous deletion of ELN leads to ELN haploinsufficiency as part of microdeletion of several genes in a small region of human chromosome 7 causing Williams syndrome (WS), a rare genetic disorder with peculiar cardiovascular phenotype including hypertension and arteriopathy manifesting as arterial stiffening, elongation, and lumen narrowing28,30,61-64. Mice with heterozygous deletion of Eln phenocopy the cardiovascular pathology of ELN haploinsufficiency as occurs in WS, including elevated blood pressure and arteriopathy with remarkable vascular lamellar disorganization, thinning, and fragmentation in both conduit and resistance arteries30,65,66. Previously, we showed by radiotelemetry that blood pressure elevation in Eln+/- mice mimics isolated systolic hypertension that is more severe in male mice and liable to further elevation by high-salt diet67. Using this mouse model of Eln haploinsufficiency combined with in vivo methods and low sodium diet, we now show that ovarian hormones moderate blood pressure elevation resulting from Eln haploinsufficiency and that the pathophysiologic mechanism of sustained systolic blood pressure elevation in female Eln+/- mice, surprisingly, involves a change in renal vasopressin receptor sensitivity and ENaC activity, leading to enhanced sodium and water retention by the kidney. The mechanisms by which the kidney maintains electrolyte and water balance are complex but key to long-term blood pressure control and are subject to regulation by several hormones, including ovarian hormones. Blood pressure modulation by ovarian hormones, mostly estrogen and progesterone, is mediated by the finetuning of tubular reabsorption of sodium and water40,68. .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted November 18, 2025. ; https://doi.org/10.1101/2025.11.18.689164doi: bioRxiv preprint Previous studies investigating the effects of ovarian hormones on renal tubular sodium and water handling have produced conflicting results. On the one hand, studies show that, during periods of elevated circulating levels of estrogen, stimulation of estrogen receptors expressed in the distal nephron (connecting tubule and collecting duct) promotes sodium and water reabsorption by increasing the expression of apical AQP2 and ENaC and basolateral AQP1; these estrogen effects facilitate extracellular fluid conservation and increased cardiac output and blood pressure11,40,69. Estrogen receptor stimulation in these nephron segments in OVX rats has also been shown to upregulate ENaC expression, an effect that could be counteracted by progesterone treatment70. These effects of estrogen would favor blood pressure elevation by promoting sodium and water reabsorption. On the other hand, other studies in OVX rats reported that long term treatment with estrogen replacement suppressed adrenal Ang II receptor expression, thereby decreasing aldosterone release and ENaC expression, thus leading to reduced blood pressure71. The findings in the current study show that OVX elevates systolic blood pressure in both Eln+/+ and Eln+/- mice, which is consistent with blood pressure lowering effect of ovarian hormones72,73. But more importantly, our study shows that, in the context of Eln haploinsufficiency, ovarian hormones dampen systolic blood pressure elevation by decreasing renal sodium and water reabsorption, at least partly, by modulating ENaC activity. Our finding is consistent with previous studies showing that estrogen has a downregulatory effect on RAS activity and ENaC expression in the kidney40,74. This effect of estrogen can lower blood pressure by decreasing sodium reabsorption in the distal nephron. Furthermore, the observed blood pressure lowering effect of ovarian hormones in female Eln+/- mice suggests a potentially additive or synergistic mechanism by which the effects of ovarian hormones and ELN (or its derivatives) in the distal nephron impinge on signaling pathways that mediate sodium reabsorption. Alternatively, the renal effects of ovarian hormones and ELN are plausibly redundant such that the blood pressure lowering effect of ovarian hormones might be a compensatory response triggered by Eln haploinsufficiency. In either mechanism, the renal effects of ELN appears to be protective or blood pressure lowering. Furthermore, we made the surprising observation that Eln haploinsufficiency suppresses the excretion of excess sodium and water following acute ECFV expansion upon blockade of vasopressin V2R, even in the absence of ovarian hormones. As widely reported and well established, the urine concentrating effect of V2R activation in the collecting duct is mediated by the trafficking and insertion AQP2 that serves as water channel in the apical membrane of epithelial principal cells, with the hypertonicity of the medullary interstitium providing the driving force for water reabsorption63,64. Interestingly, our study suggests that Eln haploinsufficiency .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted November 18, 2025. ; https://doi.org/10.1101/2025.11.18.689164doi: bioRxiv preprint augments V2R-mediated water reabsorption in acute ECFV expansion. However, the most profound effect of OVX and Eln haploinsufficiency was on the retention of excess sodium that appears to be mediated by augmented V2R activity, which is a relatively less studied but widely recognized effects of V2R signaling on renal sodium reabsorption55-57. Several lines of evidence indicate that vasopressin can induce sodium reabsorption via ENaC in the distal nephron55-58. Accordingly, a previous study by Stockand and colleagues showed that chronic V2R activation promotes apical membrane ENaC insertion in collecting duct principal cells, thus enhancing sodium reabsorption and inducing Liddle syndrome-like hypertension75. The findings in the current study are consistent with these previous observations. Indeed, subchronic treatment of female Eln+/- mice with amiloride to block ENaC after low salt diet lowered systolic blood pressure, while slightly enhancing urine flow rate and urinary sodium excretion rate. These results are consistent with the hypothesis that chronic stimulation of vasopressin receptor signaling can cause blood pressure elevation by itself or work synergically with the RAS to promote sodium and water reabsorption, and thus volume-dependent hypertension. This study sheds light on how ovarian hormones and altered sodium and water handling by the kidney are involved in hypertension associated with Eln haploinsufficiency. However, the specific mechanism by which ELN protein is physiologically involved in renal tubular function and, thus, regulation of water and electrolyte balance and long-term blood pressure control remain unexplored. It is conceivable that the modulatory effect of estrogen on vascular tone becomes more relevant in the context of elevated RAS activity as occurs in Eln insufficiency, such that depletion of ovarian hormones upon OVX further unmasks the elevated RAS activity. This is particularly plausible since Eln is not expressed in tubular epithelial cells (https://humphreyslab.com/SingleCell/displaycharts.php; https://esbl.nhlbi.nih.gov/KTEA/; https://cello.shinyapps.io/kidneycellexplorer/). Additionally, Eln haploinsufficiency might also enhance vasopressin signaling in the vasculature mediated by the low-affinity V1R to contribute to the augmented vascular tone and high blood pressure in Eln haploinsufficiency. These alternative mechanisms and hypotheses are not addressed by our study and remain to be explored. .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted November 18, 2025. ; https://doi.org/10.1101/2025.11.18.689164doi: bioRxiv preprint FUNDING INFORMATION This work was supported by NIH R01HL174004-01 and R01GM143493. DISCLOSURES The authors have no conflict of interest, financial or otherwise, to disclose. AUHTOR CONTRIBUTIONS A.J.D, I.M., G.K., J.M., and P.O. performed experiments, and processed and analyzed data; A.J.D and P.O. drafted the manuscript; J.M. and P.O. edited and revised the manuscript; and P.O. approved the final version of the manuscript. ACKNOWLEDGEMENT The authors thank Dr. Jeffrey R. Schelling for his constructive critiques of the manuscript. .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted November 18, 2025. ; https://doi.org/10.1101/2025.11.18.689164doi: bioRxiv preprint

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