Arginine vasopressin infusion is sufficient to model clinical features of preeclampsia in mice

Abstract

Copeptin, a marker of arginine vasopressin (AVP) secretion, is elevated throughout human pregnancies complicated by preeclampsia (PE), and AVP infusion throughout gestation is sufficient to induce the major phenotypes of PE in mice. Thus, we hypothesized a role for AVP in the pathogenesis of PE. AVP infusion into pregnant C57BL/6J mice resulted in hypertension, renal glomerular endotheliosis, intrauterine growth restriction, decreased placental growth factor (PGF), altered placental morphology, placental oxidative stress, and placental gene expression consistent with human PE. Interestingly, these changes occurred despite a lack of placental hypoxia or elevations in placental fms-like tyrosine kinase-1 (FLT1). Coinfusion of AVP receptor antagonists and time-restricted infusion of AVP uncovered a mid-gestational role for the AVPR1A receptor in the observed renal pathologies, versus mid- and late-gestational roles for the AVPR2 receptor in the blood pressure and fetal phenotypes. These findings demonstrate that AVP is sufficient to initiate phenotypes of PE in the absence of placental hypoxia, and indicate that AVP may mechanistically (independently, and possibly synergistically with hypoxia) contribute to the development of clinical signs of PE in specific subtypes of human PE. Additionally, they identify divergent and gestational time-specific signaling mechanisms that mediate the development of PE phenotypes in response to AVP.

Keywords: Hypertension; Mouse models; Obstetrics/gynecology; Reproductive Biology.

Figure 1. Gestational arginine vasopressin (AVP) infusion causes an isolated systolic hypertension.

Effects of pregnancy and AVP on (A and E) systolic blood pressure (SBP), (B and F) diastolic blood pressure (DBP), (C and G) pulse pressure (PP), and (D and H) heart rate (HR). Analyses by 2-way ANOVA with repeated measures and Tukey’s test for multiple comparisons. Data are expressed as the mean ± SEM. *P < 0.05. Baseline n = 10, saline n = 6, AVP n = 6.

Figure 2. Arginine vasopressin (AVP) has minor or no effect on blood flow velocity and resistance in uterine or umbilical arteries.

Effects of AVP on (A) aortic pulse wave velocity (PWV) as well as uterine artery (B) peak systolic velocity (PSV), (C) end diastolic velocity (EDV), and (D) resistive index (RI). Umbilical artery (E) PSV, (F) EDV, and (G) RI. (H) Fetal placental thickness by Doppler ultrasonography. Analyses by Student’s t test. Data are expressed as the mean ± SEM. *P < 0.05. Saline n = 11, AVP n = 13.

Figure 3. Arginine vasopressin (AVP) alters placental morphology and angiogenesis at gestational day 12.5.

(A) Effects of AVP on thickness of placental labyrinth, junctional zone, and decidua (saline n = 11, AVP n = 13). Measures of angiogenesis, including (B) spiral artery diameter and (C) spiral artery number (saline n = 11, AVP n = 13), as well as angiogenic markers placental (D) PGF and (E) FLT1 (saline n = 20, AVP n = 20). (F) HIF1A cellular localization in fractionated placentas collected from mice receiving saline or AVP (saline nuclear n = 18, chromatin n = 16; AVP nuclear n = 16, chromatin n = 14). Analyses by Student’s t test. Data are expressed as the mean ± SEM. *P < 0.05.

Figure 4. Gene expression profiling of placentas from dams infused with saline (n = 4) or arginine vasopressin (AVP) (n = 6) dams.

(A) Heatmap illustrating relative expression of genes associated with hypoxia (GRD Hypoxia gene set). (B) Heatmap illustrating relative expression of genes associated with oxidative stress (M15990 gene set). (C) Heatmap illustrating relative expression of 87 differentially expressed genes identified by DESeq2 (FDR < 0.1). (D) Venn diagrams illustrating up- and downregulated genes in AVP-infused mouse placenta (AVP Infusion) that are similarly changed in human placenta in pregnancies complicated by preeclampsia, as described in the text. Numbers indicate total number of genes significantly changed as noted in individual data sets (Leavey [ref. 5], early- or late-onset preeclampsia subsets by Tong [ref. 102], Enquobahrie [ref. 103], and Sober [ref. 103]), whereas genes noted in shared spaces of Venn diagrams are shared by at least 2 overlapping data sets.

Figure 5. Components of arginine vasopressin (AVP) system are present in placentas and trophoblasts.

(A) AVP-system components are present in human placentas. (B) Relative abundance of AVP-system components in preeclampsia compared with control pregnancies (control n = 77, preeclampsia n = 80, GSE75010). (C) Presence of AVP-system components in mouse placenta and (D) response of those components to AVP (saline n = 4, AVP n = 6). (E) Presence of AVP-system components in HTR8 human placental trophoblasts and (F) intracellular Ca²⁺ response of HTR8 cells to AVP (n = 4 distinct passages). Increases in intracellular Ca²⁺ concentration (F₃₄₀/F₃₈₀) were evoked by application of increasing concentrations of AVP. For each sample, F₃₄₀/F₃₈₀ responses for each cell were normalized to the amplitude of maximal AVP concentration. Analyses by Student’s t test. Data are expressed as the mean ± SEM. *P < 0.05.

Figure 6. Experimental design, and blood pressure effects of arginine vasopressin (AVP) and AVP-antagonist coinfusion.

Our experimental design is shown in A and described in the text. (B) Effects of saline (n = 40), AVP (n = 27), AVP + conivaptan (n = 11), AVP + relcovaptan (n = 9), and AVP + tolvaptan (n = 11) throughout pregnancy. Analyses by 3-way ANOVA followed by Tukey’s test for multiple comparisons. *P < 0.05 versus saline. (C) AVP infusion through only GD 2.5 (n = 18) and GD 9.5 (n = 12). Saline and AVP shown again for clarity. Analyses by 3-way ANOVA followed by Tukey’s test for multiple comparisons. Data are expressed as the mean ± SEM. *P < 0.05 versus AVP.

Figure 7. Renal and fetal effects of arginine vasopressin (AVP) and AVP-antagonist coinfusion.

(A) Effects of saline (n = 32), AVP (n = 30), AVP + relcovaptan (n = 15), AVP + tolvaptan (n = 9), and AVP to GD 2.5 (n = 22) and GD 9.5 (n = 12) on urine protein concentration. (B) Twenty-four–hour urine protein, (C) fetal mass, (D) placental mass, and (E) placenta/fetal mass ratio effects of saline (n = 24), AVP (n = 19), and receptor combinations (same n’s as panel A). For panels A–E, saline and AVP-treated mice are reported twice for clarity. (F) RGE with AVP, as indicated by the thickened and distorted glomerular basement membrane (pink) that was prevented by relcovaptan (n = 3 for all groups). Scale bars: 2 μm. Analyses for receptor inhibitor studies by 2-way ANOVA with Dunnett’s multiple-comparisons procedure, compared with AVP. For timing studies, analyses by 1-way ANOVA and Dunnett’s multiple-comparisons procedure, compared with saline. Data are expressed as the mean ± SEM. *P < 0.05.

Figure 8. Summary and working model.

(A) Table summarizing major phenotypes of human preeclampsia, and the sufficiency of the AVP infusion (24 ng/hr, s.c.) in pregnant wild-type C57BL/6J mice to phenocopy each, as evaluated in the current manuscript plus our recent publications (10, 104). (B) Working model, based on data presented herein, illustrating the relative involvement of AVPR1A and AVPR2 receptors at various stages of gestation, in the development of blood pressure, renal, and fetal phenotypes in the AVP infusion model.