Physiological characterization of an arginine vasopressin rat model of preeclampsia

ABSTRACT

Rodent models have contributed greatly to our understanding of preeclampsia (PE) progression in humans, however to-date no model has been able to effectively replicate the clinical presentation of the disease. This study aimed to provide a thorough physiological characterization of the arginine vasopressin (AVP)-induced rat model of PE to determine its applicability in studying the pathophysiology of PE. Female Sprague Dawley rats (n = 24) were separated into four groups (n = 6 per group) viz., pregnant AVP, pregnant saline, non-pregnant AVP, and non-pregnant saline. All animals received a continuous dose of either AVP (150 ng/h) or saline via subcutaneous mini osmotic pumps for 18 days. Full physiological characterization of the model included measuring systolic and diastolic blood pressure, and collecting urine and blood samples for biochemical analysis. AVP infusion significantly increased blood pressure and urinary protein levels in the pregnant rats (p < 0.05). Biochemical markers measured, differed significantly in the AVP-treated vs the pregnant saline groups (p < 0.05). Placental and individual pup weight decreased significantly in the pregnant AVP vs pregnant saline group (p < 0.05). The physiological and hematological data confirm the usefulness of this rat model in the study of PE, since AVP-induced vasoconstriction increases peripheral resistance and successfully mimics the pathological changes associated with PE development in humans.

Abbreviations: PE: preeclampsia; AVP: arginine vasopressin; ISSHP: International Society for the Study of Hypertension in Pregnancy; ACOG: American College of Obstetricians and Gynecologists; RUPP: reduced uterine perfusion pressure; sFlt-1: soluble fms-like tyrosine kinase; VEGF: vascular endothelial growth factor; PlGF: placental growth factor; AVP: arginine vasopressin; PAVP: pregnant AVP-treated; PS: pregnant saline; GD: gestational day; ALT: alanine transaminase; NAVP: non-pregnant AVP-treated; NS: non-pregnant saline; AST: aspartate aminotransferase; HDL: high-density lipoprotein; RBC: red blood cell; RAAS: renin-angiotensin aldosterone system; HELLP: hemolysis, elevated liver enzymes, low platelet.

KEYWORDS:

• Arginine vasopressin
• blood pressure
• preeclampsia
• pregnancy
• proteinuria

Introduction

Preeclampsia (PE) is a multifactorial, multi-organ hypertensive disorder manifesting after 20 weeks of gestation, impacting 5–8% of all pregnancies, making it the leading cause of maternal and fetal morbidity and mortality globally (Marshall et al. 2018). Despite advancements in understanding the pathophysiology of PE, its early diagnosis remains a challenge. Initially, PE was characterized by de novo hypertension coupled with proteinuria (Brown et al. 2018) however, the International Society for the Study of Hypertension in Pregnancy (ISSHP) and the American College of Obstetricians and Gynecologists (ACOG) have redefined PE, stating that proteinuria is not mandatory for PE diagnosis and allowing for the inclusion of cases that exhibit signs of renal, hepatic, and/or hematological pathologies (Brown et al. 2018).

Treatment and management of PE is hindered by late clinical presentation often warranting premature fetal delivery. Ethical consideration and large enough sample numbers make it difficult to study PE in humans (Sones and Davisson 2016), which has resulted in a shift toward the use of animal models. Various animal models have contributed greatly to our understanding of PE. These including the reduced uterine perfusion pressure (RUPP) (Li et al. 2012), soluble fms-like tyrosine kinase (sFlt-1) rodent model (Maynard et al. 2003) and BPH/5 mouse models (Davisson et al. 2002) have been widely used to study the disorder. However, these models are limited in that the RUPP and sFlt-1 models are unable to reproduce liver dysfunction and intrauterine growth restriction associated with PE development (Maynard et al. 2003; Li et al. 2012), while the BPH/5 model indicates a preexisting hypertension in non-pregnant mice (Davisson et al. 2002). The recent data demonstrate the effectiveness of some models in treating PE symptoms, for example; aspirin improved maternal and fetal symptoms in a preclinical model of PE (Li et al. 2018; Zhang et al. 2018). Moreover, vasodilators such as sildenafil ameliorates maternal phenotype in PE models, however, its fetal and placental effects are inconsistent (Motta et al. 2015; Gillis et al. 2016). Also, proangiogenic vascular endothelial growth factor (VEGF) and placental growth factor (PlGF), improve PE phenotypes in soluble fms-like tyrosine kinase-1 (sFlt1)-induced preeclampsia (PE) rodent models (Spradley et al. 2016; Zhu et al. 2016). Despite the plethora of evidence, not all aspects of PE are fully understood. Therefore, it is essential that alternative animal models are available to further develop the study of PE pathogenesis.

Arginine vasopressin (AVP), a vasoconstrictor and antidiuretic hormone was recently used in the development of a murine PE model, which displayed all of the characteristic phenotypes of PE (Santillan et al. 2014). AVP plays a central role in the mechanisms related to PE development viz., vascular function, angiogenesis, renal dysregulation, and immunological abnormalities (Santillan et al. 2014). In non-pregnant states, AVP exerts its physiological effects via V1a receptors, inducing vasoconstriction and regulating neuronal and immune cell function (Koshimizu et al. 2012), as well as modulating angiogenesis in the hypothalamus (Alonso et al. 2008). It promotes water reabsorption in the kidney via V2 receptors (Guelinckx et al. 2016), thereby regulating blood pressure and blood volume (Koshimizu et al. 2012; Santillan et al. 2014). It also supports the regulation of the hypothalamic-pituitary-adrenal axis via V1b receptors, which indirectly alters immune, vascular, and renal functions (Buchwalter et al. 2008; Koshimizu et al. 2012). AVP is also involved in T helper 1 cell predominance and activation of dendritic and T helper 17 cells associated with PE development (Elenkov and Chrousos 2006; Russell and Walley 2010). Hence, the normal physiological role of AVP is similar to the mechanisms underlying the vascular, immune, renal, and angiogenic dysfunction associated with PE (Sandgren et al. 2015), warranting its investigation in PE development.

Despite the murine AVP model of PE producing symptoms similar to those observed clinically, this model has yet to be replicated in rats, a prototype which is preferred for pregnancy-specific disorders due to a similar placental development observed in humans (Soares et al. 2014). In a previous study conducted by our lab, we showed significantly increased serum levels of transforming growth factor beta-2 and a significant elevation in the activity of the liver injury enzymes arginase and 5ʹ-nucleotidase suggestive of AVP-induced injury (Govender et al. 2021). Therefore, this study aimed to characterize the physiological, hematological, and biochemical changes associated with an AVP-induced rat model of PE, in order to determine its applicability in studying the pathophysiology of human PE.

Results

Preliminary dose-response study

The physiological dose-response outcomes supported the use of an AVP dose of 150 ng/h (Figure 1). Lower AVP dosages (50 ng/h and 100 ng/h) were unable to significantly elevate blood pressure and proteinuria nor did it reduce placental and pup weight. Pregnant rats treated with AVP (150 ng/h) showed significantly increased systolic blood pressure and urinary protein levels and reduced individual pup and placental weights, respectively (Figure 1).

Figure 1. Effect of chronic AVP infusion (50, 100 and 150 ng/h) on (A) systolic blood pressure, (B) urinary protein concentration and (C-F) fetal and placental parameters in pregnant Sprague-Dawley rats. (A) Infusion of 150 ng/h AVP produced a larger increase in systolic blood pressure in pregnant rats than lower doses (50 and 100 ng/h). (B) Rats infused with 150 ng/h AVP showed a marked increase in urine protein levels in comparison to lower doses (50 and 100 ng/h). (C) Individual placental weight, AVP-infusion reduces placental weight. (D) Total number of pups, AVP-infusion does not induce a great change in the total number of pups produced. (E) 150 ng/h AVP infusion induced a significant reduction in total fetal weight in comparison to 50 ng/h. (f) Individual pup weight was significantly reduced pups produced from rats infused with 150 ng/h AVP in comparison to rats infused with 50 ng/h AVP. One-way ANOVA analyses followed by Tukey’s multiple comparison analyses. Data is shown as mean ± SD, *p < 0.05.

Experimental study

Statistically significant elevations were noted for both systolic [143.00 (139.67–144.00) mmHg vs 125.00 (124.00–127.00) mmHg; p < 0.05] and diastolic [103.50 (100.33–106.88) mmHg vs 81.00 (80.00–87.00) mmHg; p < 0.001] blood pressure throughout pregnancy in the pregnant AVP-treated (PAVP) vs the pregnant saline (PS) group (Figure 2A-B). The urine protein: creatinine ratio was increased in the PAVP compared to the pregnant saline group on gestational day (GD) 8 [PAVP: 0.79 (0.73–0.88) g/day vs PS: 0.49 (0.43–0.49) g/day; p = 0.006] and GD 14 [PAVP: 0.84 (0.74–0.94) g/day vs PS: 0.41 (0.39–0.44) g/day; p = 0.0002; Figure 2E]. Water intake decreased in the PAVP in comparison to the PS group on GD 8 and GD 14 (Figure 2C), whereas urinary output decreased significantly in PAVP rats [17.00 (12.00–20.00) ml] compared to the PS rats [25.50 (25.00–29.00) ml; p < 0.05; Figure 2D].

Figure 2. Clinical parameters (A-B) systolic and diastolic blood pressures (mmHg); (C) water intake (ml); (D) urinary output (ml) and (E) urine protein:creatinine ratio levels (mg/dL) across all study groups. Systolic (A) and diastolic (B) blood pressure was significantly increased in AVP-treated groups in comparison to saline groups. (C) Water intake is significantly upregulated in the pregnant groups in comparison to the non-pregnant rat groups. (D) Urine output is significantly reduced in the pregnant AVP-treated group as compared to the pregnant saline group (E) AVP infusion in pregnant rats induced significant elevations in urine protein: creatinine ratio levels in comparison to saline infusion on GD 8 and 14. Kruskal-Wallis and Dunn’s post hoc multiple comparison tests were used to compare the medians between groups. Data is shown as median and interquartile range, *p < 0.05, **p < 0.001. # Data for non-pregnant (NS and NAVP) groups were within normal ranges and are not illustrated.

The hematology results show significantly higher serum alanine transaminase (ALT) levels in the PAVP [58.50 (57.00–60.00) IU/L] compared to the non-pregnant AVP-treated (NAVP) [40.00 (38.00–44.00) IU/L; p < 0.001] and saline-treated groups [non-pregnant saline (NS): 51.50 (46.00–54.00) IU/L; PS: 48.00 (45.00–49.00) IU/L; p < 0.05], with liver weight also significantly higher in the former group [12.39 (11.99–13.40) g; p < 0.001; Figure 3B & G]. Conversely, aspartate aminotransferase (AST) levels were significantly down-regulated in the PAVP [131.00 (130.00–135.00) IU/L] compared to both the NS [149.00 (143.50–150.00) IU/L] and NAVP [143.50 (140.00–148.00) IU/L; p < 0.05; Figure 3H] groups. A non-significant increase in AST levels was noted in the PAVP group in comparison to the PS group [129.50 (127.00–133.00) IU/L; p > 0.05; Figure 3H]. Despite down-regulated lactate dehydrogenase (LDH) levels in the PAVP [1300.00 (1176.50–1359.50) IU/L] vs the NAVP group [1431.00 (1400.00–1738.00) IU/L; p < 0.05; Figure 3I], both AVP-treated groups displayed significantly higher LDH levels when compared to the saline-treated groups [NS: 570.00 (487.00–761.50) IU/L; PS 538.00 (498.00–549.00) IU/L; p < 0.05].

Figure 3. Metabolic parameters (A) weight (g) and (B) liver weight (g); and biochemical parameters (C) alanine aminotransferase (ALT, mmol/L); (D) aspartate aminotransferase (AST, mmol/L); (E) lactic acid dehydrogenase (LDH, mmol/L); (F) total cholesterol (mmol/L); (G) HDL cholesterol (mmol/L); (H) triglycerides (mmol/L) and (I) blood glucose (mmol/L), measured across all study groups on GD 18. (A) Pregnant rat groups demonstrate significant elevations in weight in comparison to the non-pregnant control groups. (B) Liver weight is increased in the PAVP group in comparison to the PS group. (C) ALT concentrations were significantly elevated in the PAVP vs the PS group. (D) AST concentrations are elevated in the PAVP group in comparison to the PS group. (E) LDH levels are significantly increased in the PAVP group in comparison to the PS group. (F) Total cholesterol and (G) HDL cholesterol is significantly reduced in the PAVP group in comparison to the PS group, while (H) Triglyceride levels in the PAVP group are significantly elevated as compared to the PS group. (I) Blood glucose levels are higher in the PAVP group when compared to the PS group on GD 9, conversely the PAVP group is reduced in comparison to the PS group on GD 18. Kruskal-Wallis and Dunn’s post hoc multiple comparison tests were used to compare the medians between groups. Data is shown as median and interquartile range, *p < 0.05, **p < 0.001. # Data for non-pregnant (NS and NAVP) groups were within normal ranges and are not illustrated.

Cholesterol and high-density lipoprotein (HDL) levels were significantly decreased in the PAVP [cholesterol: 1.10 (1.10–1.30) mmol/L; HDL: 0.65 (0.64–0.68) mmol/L] and NAVP groups [cholesterol: 1.20 (1.00–1.20) mmol/L; HDL:0.77 (0.70–0.79) mmol/L] vs the PS group [cholesterol: 1.29 (1.25–1.32) mmol/L; HDL: 0.94 (0.91–0.96) mmol/L; p < 0.05; Figure 3D–E]. Triglyceride concentrations increased significantly in the PAVP [2.69 (2.16–3.05) mmol/L] compared to the NAVP [0.64 (0.58–0.69) mmol/L], NS [0.84 (0.47–1.09) mmol/L] and PS [1.27 (0.98–1.30) mmol/L] groups (p < 0.05; Figure 3F).

Blood glucose levels decreased significantly in the PAVP [4.35 (3.90–4.50) mmol/L] group in comparison with the NAVP [5.65 (5.40–6.40) mmol/L] and NS [6.30 (6.05–6.50) mmol/L] groups on GD18 (p < 0.05; Figure 3C). Non-significant reductions were noted for red blood cell (RBC) count, platelets, and hematocrit levels in the PAVP group compared to the NAVP group (p > 0.05; Table 1), the levels were above the normal reference range.

Table 1. Hematological parameters between the non-pregnant AVP and pregnant AVP groups on GD 18. 

	Rat Group		p value
	Non-pregnant AVP (n = 6)	Pregnant AVP (n = 6)
Complete Blood Count
Red Cell Count (10^12/L)	8.00 (6.20–8.44)	7.10 (6.23–8.73)	0.909
MCV (fL)	63.10 (60.90–65.00)	64.20 (61.40–64.20)	0.909
RDW (%)	14.70 (13.10–15.90)	13.20 (12.50–16.30)	0.732
Hemoglobin (g/dL)	14.90 (12.00–15.90)	13.40 (12.60–14.20)	0.423
MCHC (g/dL)	29.60 (29.20–29.90)	29.30 (28.90–29.80)	0.424
MCH (pg)	19.00 (18.40–19.40)	18.80 (17.80–19.20)	0.566
White Cell Count (10^9/L)	4.72 (2.57–6.14)	5.07 (2.52–6.17)	0.909
Basophil count (10^9/L)	0.01 (0.00–0.01)	0.01 (0.00–0.01)	1.00
Eosinophil count (10^9/L)	0.11 (0.03–0.21)	0.10 (0.04–0.13)	0.732
Lymphocyte count (10^9/L)	3.33 (1.74–4.18)	4.03 (1.46–4.31)	0.569
Monocyte count (10^9/L)	0.26 (0.09–0.56)	0.28 (0.16–0.29)	0.909
Neutrophil count (10^9/L)	0.71 (0.71–1.38)	0.74 (0.49–1.71)	0.729
Hematocrit (%)	51.20 (46.20–53.00)	49.70 (48.10–52.30)	0.871
Platelets (10^9/L)	916.00 (856.00–973.00)	908.00 (895.00–929.00)	1.00

* p < 0.05 was considered statistically significant

Note: Data are presented as median (25^th percentile – 75^th percentile)

# saline hematological control data were within normal range

A significant reduction in the serum concentrations of sodium [139.00 (138.00–143.00) mmol/L vs 143.00 (141.00–143.00) mmol/L]; urea [4.50 (3.50–5.80) mmol/L vs 5.60 (5.50–6.00) mmol/L]; and chloride [98.00 (97.00–100.00) mmol/L vs 105.00 (104.00–106.00) mmol/L] was noted between the PAVP group compared to the NAVP group (p < 0.05; Table 2). Potassium levels decreased significantly in the PAVP [4.55 (3.90–4.80) mmol/L] and NAVP [4.40 (4.20–4.80) mmol/L] vs the NS group [5.00 (4.80–5.40) mmol/L; p < 0.05; Table 2]. A reduction in creatinine levels was observed in both AVP groups [NAVP: 29.00 (27.00–33.00) µmol/L; PAVP: 28.50 (24.00–30.00) µmol/L; p < 0.05; Table 2].

Table 2. Serum electrolyte levels across experimental groups on GD 18. 

	Rat Group				p value
	Non-pregnant Saline (n = 6)	Non-pregnant AVP (n = 6)	Pregnant Saline (n = 6)	Pregnant AVP (n = 6)
Calcium (mmol/L)	2.42 (2.36–2.45)	2.36 (2.34–2.40) ^d	2.41 (2.40–2.42)	2.36 (2.35–2.36) ^c g	0.076
Chloride (mmol/L)	103.00 (100.00–104.00)	105.00 (104.00–106.00) ^a d	103.00 (99.00–104.00)	98.00 (97.00–100.00) ^f	0.012*
Creatinine (µmol/L)	35.50 (33.50–38.00)	29.00 (27.00–33.00) ^a	29.50 (27.00–34.00) ^b	28.50 (24.00–30.00) ^c	0.049*
Potassium (mmol/L)	5.00 (4.80–5.40)	4.40 (4.20–4.80) ^a	4.40 (4.20–4.70) ^b	4.55 (3.90–4.80) ^c	0.018*
Sodium (mmol/L)	140.00 (139.00–141.00)	143.00 (141.00–143.00) ^a	141.50 (139.00–145.00)	139.00 (138.00–143.00) ^e	0.113
Urea (mmol/L)	5.70 (5.45–5.95)	5.60 (5.50–6.00) ^d	4.50 (4.30–4.90) ^b	4.50 (3.50–5.80) ^c e	0.046*

* p < 0.05 was considered statistically significant

NS vs NAVP: ^a p < 0.05; NS vs PS: ^b p < 0.05; NS vs PAVP: ^c p < 0.05; NAVP vs PS: ^d p < 0.05; NAVP vs PAVP: ^e p < 0.05, ^f p < 0.001; PS vs PAVP: ^g p < 0.05

Note: Data are presented as median (25^th percentile – 75^th percentile)

Pup numbers increased significantly in the PAVP [12.00 (11.00–12.00)] vs PS groups [9.00 (8.00–10.00); p < 0.05; Table 3], however, the weight per pup was significantly lower in the PAVP [1.57 (1.45–1.67) g vs the PS groups [1.79 (1.70–1.85) g; p < 0.05]. We also demonstrate significantly lower individual placental weights in the PAVP [0.39 (0.31–0.46) g] vs the PS groups [0.59 (0.55–0.63) g; p < 0.05; Table 3].

Table 3. Fetal and placental parameters. 

	Rat Group		p value
	Pregnant Saline (n = 6)	Pregnant AVP (n = 6)
Total Placenta weight (g)	5.42 (4.68–5.94)	4.00 (3.38–5.16)*	0.04
Individual placenta weight (g)	0.59 (0.55–0.63)	0.39 (0.31–0.46)*	0.002
Individual Pup weight (g)	1.79 (1.70–1.85)	1.57 (1.45–1.67)*	0.02
Number of pups	9.00 (8.00–10.00)	12.00 (11.00–12.00)*	0.04

Note: Data are presented as median (25^th percentile – 75^th percentile)

* p < 0.05 was considered statistically significant

Histological evaluation of liver, kidney and placenta

The histological examination of the liver in both the untreated and AVP treated groups appeared normal. In the kidney, mild mesangial cell increase together with an increase in mesangium (Figure 4A-B) was noted in the AVP-treated (NAVP and PAVP) in contrast to the untreated groups. Mild focal cellular glomerular crescents with concurrent reduction in the Bowmans space was also evident (Figure 4A-B). In the placenta, the labyrinth trophoblast cells lining the vascular channels appeared normal with occasional vacuolation while the spongiotrophoblasts were frequently degenerative in the AVP-treated group vs the untreated group (Figure 4C-F). The spongiotrophoblasts were often necrotic, with phagocytic material visible (Figure 4E-F).

Figure 4. Histopathological examination of tissue sections from pregnant AVP treated rats (H&E stain) displaying (from top left plate clockwise; red arrows): (A-B: Kidney): mild mesangial cell increase, increase in mesangium and reduction in the Bowmans space, 40x magnification; (C-F: Placenta): occasional vacuolation along labyrinth trophoblast cells, 20x magnification; degenerative spongiotrophoblasts, 40x magnification; red blood cell phagocytosis, 40x; and fibrinoids, 40x respectively.

Discussion

This study demonstrates that AVP infusion (150 ng/h) resulted in significantly elevated blood pressure and urinary protein, features that typifies PE. Both systolic and diastolic blood pressure were significantly elevated throughout pregnancy in the PAVP vs the PS group. Similarly, chronic AVP infusion of 24 ng/h in a murine model demonstrated increased systolic blood pressure and negatively impacted fetal growth and development (Santillan et al. 2014). The short biological half-life of AVP prevents the direct measurement of secreted AVP (Santillan et al. 2014), hence, copeptin, a stable protein by-product of AVP synthesis released in a 1:1 ratio with AVP, is the substitute biomarker for measuring AVP secretion (Dobsa and Edozien 2013; Sandgren et al. 2015). In humans, higher plasma and serum copeptin levels in the preeclamptic compared to normotensive pregnancies is indicative of higher AVP levels as well [(serum: normotensive: 1.69 ± 1.02 ng/mL vs PE: 2.27 ± 2.00 ng/mL) (Yeung et al. 2014) and (plasma: normotensive: 0.31 ± 0.09 ng/mL vs mild PE: 0.62 ± 0.16 ng/mL) (Zulfikaroglu et al. 2011)].

Earlier studies also linked AVP to arterial blood pressure regulation (Jablonskis and Howe 1993; Song and Martin 2006; Li et al. 2012). The elevations in both systolic and diastolic blood pressure in our study throughout pregnancy in the PAVP group, suggests that arginine vasopressin stimulates the renin-angiotensin aldosterone system (RAAS). This results in vasoconstriction, which is mediated via the V1a receptor and consequently increases peripheral resistance and systemic blood pressure as observed in our study (Qian 2018). Myocardial atrial contraction results in an atrial-induced increase in end-diastolic pressure, which subsequently enhances ventricular contraction. Arginine vasopressin increases the impact of norepinephrine and Ang II on cardiac muscle and blood vessels thus altering hemodynamic function (Lee et al. 2003), and negatively affects myocardial contraction (Goldsmith 2005; Goldsmith and Gheorghiade 2005). Chronic hypertension results in diastolic dysfunction and consequent left ventricular hypertrophy thereby reducing cardiac compliance (Lorell and Carabello 2000). This results in a higher diastolic pressure–volume relationship where even minor elevations in left ventricular end-diastolic volume induces a significant rise in left ventricular end diastolic pressure (Gutierrez and Blanchard 2004). The pronounced effect of AVP on diastolic pressure may be due to the exaggerated interaction of AVP with the V1A and V2 receptors on peripheral blood vessels (Goldsmith 2005; Goldsmith and Gheorghiade 2005).

Despite the exclusion of proteinuria as a characterizing factor (ACOG 2013), it remains significant in PE diagnosis (Guida et al. 2018; Özkara et al. 2018; Tanacan et al. 2019). We report significantly increased urinary protein: creatinine ratios in the PAVP group compared to the pregnant saline group on GD 8 and 14. It is possible that AVP affects the control of glomerular filtration rate via the macula densa and the tubulo-glomerular feedback mechanism as a result of an increase in intraglomerular pressure. The macula densa cells detects the increased tubular ion content subsequently activating the RAAS, which inhibits the juxtaglomerular release of nitric oxide. This results in arteriolar vasoconstriction and increases glomerular and systemic blood pressure, causing glomerular hyperfiltration and proteinuria. A similar effect was previously demonstrated in diabetic rats (Bankir et al. 2001; Bolignano and Zoccali 2010).

Likewise, chronic AVP administration in pregnant mice induces glomerular endotheliosis and consequent proteinuria (Sandgren et al. 2018). In a nitric oxide synthase inhibition rat PE model, elevations in systolic blood pressure were associated with increased glomerular pressure and consequent glomerular damage and proteinuria (Baijnath et al. 2014). Shedding of the podocyte barrier due to the intermittent separation of the foot processes from the basement membrane may result in proteinuria (Koop et al. 2003). An angiogenic balance between vascular endothelial growth factor (VEGF) and its receptor sFlt-1 maintains the integrity and function of glomerular filtration barrier (al Embarazo et al. 2007). In PE, increased levels of sFlt-1, an antagonist of the VEGF receptor, offsets this balance by inhibiting its receptor binding and reducing VEGF levels (Müller-Deile and Schiffer 2011; Govender et al. 2012), negatively impacting the glomerular structural integrity resulting in proteinuria (Baijnath et al. 2017). Moreover, elevations in urinary mRNA levels of podocin and nephrin in early onset PE suggest glomerular damage and podocyte loss characteristic of PE development (Baijnath et al. 2017).

Glomerular damage increases glomerular permeability, thereby permitting entry of larger-sized proteins into the filtrate. The increase in peripheral vascular resistance due to AVP treatment, elevates systemic BP with consequent glomerular injury (Santillan et al. 2014) as demonstrated by the higher urinary protein levels observed in the AVP-treated rats in our study. Kidney histology as shown in Figure 4 highlights the effects of mild increase in mesangium, mild glomerular crescents and reduction in the Bowman’s space in AVP-treated rats (NAVP and PAVP). The Bowman’s capsular space protects glomerular function by acting as a shield against leukocyte infiltration (Chen et al. 2018). Glomerular epithelial crescents produced by the aggregation of inflammatory cells and proliferating epithelial cells in the Bowman’s space, are characteristic of glomerulonephritis (D’Souza et al. 2013). Thus, our data suggest an AVP-induced leukocyte infiltration and accompanying glomerular damage may contribute to increased urinary protein levels in the AVP- treated groups. In our study, water intake increased proportionally with gestational days amongst the pregnant (PAVP and PS) and NAVP groups, while urinary output reduced significantly in PAVP rats compared to the PS rats. Reductions in urinary output may be attributed to the antidiuretic effect of AVP, via V2 receptor activation and increased expression of aquaporin-2 channels resulting in water retention and reduced urinary output (Guelinckx et al. 2016).

In our study, we notice a significant reduction in the serum concentrations of sodium, urea, and chloride was noted between the PAVP group compared to the NAVP group. Potassium levels decreased significantly in the PAVP and NAVP vs the NS group. Sodium, chloride, and creatinine levels in the PAVP group showed a lower trend in comparison with the PS group, however, these differences were not significant. Reduced serum sodium and potassium levels suggest a dysregulation in their transport across the vascular smooth-muscle cell membrane (Indumati et al. 2011). Sodium reabsorption mediated by AVP within renal tubules, creates a salt-sensitive rise in blood pressure while urea retention may increase plasma urea levels, indirectly increasing glomerular filtration rate (Qian 2018). Our results corroborate others (Anastasio et al. 2001; Bankir et al. 2017), and supports a direct relationship between increased GFR and urine osmolality in both humans and rats. Elevated serum chloride levels in PE may possibly inhibit vessel dilation due to increased osmolality (Barrett et al. 2010).

The observed reduction in creatinine levels in both AVP groups contradicts other reports of elevated creatinine levels in PE (Manjareeka and Nanda 2013; Patil et al. 2016). However, creatinine levels may be influenced by many other factors including the glomerular filtration rate and differences in filtration fraction (Huang et al. 2011), since higher filtration fractions reduce efferent blood flow resulting in decreased tubular creatinine secretion. Moreover, vasoconstriction of the efferent arteriole may be due to increased RAAS activation induced by AVP, which increases angiotensin II that significantly influences serum creatinine levels.

In addition, we also investigated serum biochemical parameters in the AVP model of PE. We observed significantly higher serum ALT levels in the PAVP compared to the NAVP and saline-treated groups, with liver weight also significantly higher in the former group. Conversely, AST levels were significantly down-regulated in the PAVP compared to both the NS and NAVP groups. A non-significant increase in AST levels was noted in the PAVP group in comparison with the PS group. Despite the noticeable dysregulation of transaminase levels in our study, the liver histology of the PS and PAVP groups was normal, suggesting that while a 150 ng/h infusion of AVP could elevate the level of liver enzymes, it was not high enough to cause liver damage of hemolysis, elevated liver enzymes, low platelet (HELLP) associated with PE. Despite down-regulated LDH levels in the PAVP vs the NAVP group, both AVP-treated groups displayed significantly higher LDH levels when compared to the saline-treated groups.

Liver diseases are reported to affect approximately 3% of pregnancies, resulting in maternal and fetal mortality (Mikolasevic et al. 2018), hence it is important to not rule out these variations in the liver injury enzymes. It may be potential indicators of liver dysfunction such as intrahepatic cholestasis of pregnancy or acute fatty liver of pregnancy manifesting in late pregnancy (Mikolasevic et al. 2018). This is typical in patients who present with HELLP syndrome, which is seen in severe cases of PE, however, our data indicates mild AVP induced blood pressure elevations, suggestive of mild PE onset. HELLP syndrome is a pregnancy-associated liver disease and is a predisposing factor for the progression of PE to eclampsia (Barton and Sibai 2004; Jeyabalan 2013; Brown et al. 2018). Reproducing this HELLP phenotype in animal models of PE will support the investigation of mechanisms implicated in PE development and its progression from severe PE to eclampsia.

We recently reported higher serum levels of liver injury enzymes arginase, and 5ʹ-nucleotidase in AVP-treated pregnant rats in comparison with saline-treated rats (Govender et al. 2021). Higher 5ʹ-nucleotidase levels observed in the PAVP group is suggestive of possible hepatotoxicity and hepatobiliary disease as previously reported (Dixon and Purdom 1954; Carakostas et al. 1990). The observed increase in these enzyme levels are indicative of liver injury induced by AVP, however, the extent of the liver injury displayed in this model was insufficient to produce the hepatic dysfunction associated with the HELLP syndrome (Govender et al. 2021). Our results are in accordance with Sandgren’s group, who reported similar findings (Sandgren et al. 2018). The abnormal liver markers and elevated transaminase levels observed in our study may be indicative of acute pregnancy-initiated liver dysfunction.

There is limited data associated with the AVP-induced rat PE model. Nonetheless, higher serum ALT, AST, LDH, and aminotransferase levels occur in PE compared to healthy pregnant women (Dacaj et al. 2016; Gupta et al. 2019). Chronic AVP possibly creates a hypoxic environment that alters liver function and increases serum AST and ALT levels. Additionally, endothelial dysfunction, a pathognomonic PE feature may alter the prostacyclin: thromboxane ratio with consequent elevations in thromboxane causing hepato-vascular vasoconstriction (Dacaj et al. 2016). Moreover, hypoxia in PE may increase glycolysis consequently increasing LDH activity (Lu et al. 2005), indicating cellular damage and their potential use to evaluate disease severity (Qublan et al. 2005).

Our data also highlight a reduction in cholesterol and HDL levels in the PAVP and NAVP groups in comparison with the PS group. Reduced HDL levels in our study may be linked to the endothelial dysfunction as previously reported (ACOG 2013; White et al. 2019). However, reduced cholesterol levels in AVP-treated rats in comparison with pregnant saline rats contradicts others (Gohil et al. 2011; White et al. 2019), which indicate elevated cholesterol levels in PE. Triglyceride concentrations increased significantly in the PAVP compared to the NAVP- and saline-treated groups. Maternal serum triglyceride levels increase during pregnancy to accommodate the fetal nutritional needs (Cortés-Vásquez et al. 2018), however, an excess in triglyceride levels are associated with PE development (Huang et al. 2013). Arginine vasopressin exerts an anti-lipolytic effect via V1a receptors through the down-regulation of tissue lipase, thereby inhibiting triglyceride breakdown (Hiroyama et al. 2007). Elevations in triglyceride levels may interfere with placental vascular development resulting in inadequate implantation and/or poor placental perfusion (Mayret-Mesquiti et al. 2007; Vrijkotte et al. 2012; El Khouly et al. 2016). Additionally, increased triglyceride levels may promote oxidative stress via peroxidation of placental lipids and trophoblasts resulting in endothelial cell damage (Niromanesh et al. 2012; El Khouly et al. 2016). Others also demonstrated higher triglyceride levels in PE compared to normotensive pregnancies (Lima et al. 2011; Huang et al. 2013; Saha et al. 2013), suggestive that triglyceride elevation may be linked to spiral artery atherosclerosis/thrombosis and consequent endothelial dysfunction (Gilbert et al. 2008; Saha et al. 2013).

Blood glucose levels decreased significantly in the PAVP group in comparison with the NAVP and NS groups on GD18. Acute AVP infusion in rats produces a temporary rise in blood glucose concentration (Hems et al. 1975; Taveau et al. 2016), however, the reductions noted in our AVP- treated rats suggest that AVP most probably inhibited gluconeogenesis and glycogenolysis, a process that warrants further investigation.

Despite the non-significant reductions noted for RBC count, platelets, and hematocrit levels in the PAVP group compared to the NAVP group, the levels were above normal. Notably, AVP induces the proliferation and differentiation of red blood cell precursors (Mayer et al. 2017), accounting for the elevation noted for RBC counts and hematocrit. Increased hematocrit and RBC levels increase blood viscosity and peripheral resistance with consequent elevations in blood pressure (Emamian et al. 2017). Decreased platelet count is associated with the development of the HELLP syndrome in severe PE, which is characterized by hemolysis, elevated liver enzyme, and low platelet count (Weiner et al. 2016). High platelet levels observed in the AVP-treated groups are likely due to the mild AVP-induced elevations in blood pressure (120–140 mmHg), which was unable to excessively alter platelet levels. Interestingly, our findings are corroborated by others, who also reported no significant changes to hematological parameters between PE and normal pregnant women (Makuyana et al. 2002; Hershkovitz et al. 2005; Siddiqui et al. 2011).

Despite a significant increase in pup numbers in the PAVP vs PS group, the weight per pup was significantly lower in the PAVP in contrast to the PS group. Abnormal placentation characteristic of preeclamptic placentae leads to reduced placental perfusion and hypoxia (Cheng and Wang 2009) with consequent fetal growth restriction (Cotechini et al. 2014). We also demonstrate significantly lower individual placental weights in the PAVP vs the PS groups. The smaller placentae may be associated with reduced uteroplacental blood flow due to vasoconstriction of spiral arterioles, which directly influences fetal growth (Ferrazzani et al. 2011), mirroring human PE. Placental spongiotrophoblasts are responsible for increasing nutrient availability to the placenta in response to the maternal metabolic adjustments to pregnancy (Hu and Cross 2009; Eaton et al. 2020). Our histology data demonstrate that the labyrinth trophoblast cells lining the vascular channels appeared normal with occasional vacuolation while the spongiotrophoblasts were frequently degenerative in the AVP-treated group vs the untreated group. The spongiotrophoblasts were often necrotic, with phagocytic material visible, which correlates with the reduced relative size of the basal/junctional zone. It is possible that AVP-induced necrosis and cellular degeneration leading to decreased spongiotrophoblast proliferation and inadequate placental development on the fetal side, which corresponds to the lower individual pup weights observed in the treated groups (Eaton et al. 2020).

Despite higher pup numbers observed in the PAVP group, the weight per pup was smaller, indicative of restricted fetal development in comparison to the PS group. The placenta is integral in transporting glucose from the maternal to fetal circulation (Furukawa et al. 2011). The AVP-induced glucose reduction and placental impairment observed in our study may have consequently altered glucose transport to the fetus, which correlates with the lower pup weights shown in the AVP- treated groups. Similar findings are reported by various others (Crews et al. 2000; Li et al. 2012; Macdonald-Wallis et al. 2014; Santillan et al. 2014). Likewise, lower birth weight and intrauterine growth restriction were reported in C57BL/6 J pregnant mice chronically infused with AVP (Santillan et al. 2014). Similarly, in a RUPP rat PE model, uteroplacental perfusion was reduced by almost 40% in vivo, resulting in late gestational hypertension, proteinuria, smaller litter sizes, and fetal growth restriction (Crews et al. 2000; Li et al. 2012). Elevated maternal blood pressure occurring early in pregnancy is thus associated with reduced birth weight and small-for-gestational-age babies (Macdonald-Wallis et al. 2014). However, an earlier study reported that preeclampsia is associated with both pregnancies with normal placental function as well as those with abnormal placentation and intrauterine growth restriction (Rasmussen and Irgens 2003). Late-onset PE pregnancies (>34 weeks) are usually linked to normal birth weights (Odegård et al. 2000), whereas in early-onset PE pregnancies, the presence or absence of fetal growth restriction did not induce significant differences in the degree of hypertension or maternal complications (Haddad et al. 2007).

To our knowledge, this is the first study to successfully replicate an AVP-induced mouse PE model to a Sprague-Dawley rat model. We demonstrate significantly increased systolic and diastolic blood pressures and proteinuria in the AVP-treated rats in comparison with the untreated rats. The physiological and hematological data confirm that this AVP-induced rat model has the potential to adequately reproduce the clinical PE features of elevated blood pressure, proteinuria, fetal growth restriction together with elevations in several blood and lipid parameters. We demonstrate significantly higher serum levels of ALT and triglycerides along with reduced HDL levels in the AVP-treated pregnant group, confirming the potential role of AVP in the development of PE.

Materials and methods

Animal welfare

Sprague-Dawley rats aged 10–12 weeks (160–180 g) were housed in polycarbonate cages under standard laboratory conditions of temperature (22 to 24°C), humidity (60%) and illumination (12 h light/dark cycles) with ad libitum access to standard rat chow (Meadows Feeds, Pietermaritzburg, South Africa) and normal drinking water.

Preliminary dose-response study

A pilot dose-response study used three AVP doses (50, 100, and 150 ng/h) to determine the optimum and safe dose required to produce the clinical symptoms of PE. Six pregnant female Sprague-Dawley rats (n = 2 per dose) were surgically implanted with ALZET mini-osmotic pumps (model 2004; Durect Corporation, Cupertino, CA), on GD 1 to subcutaneously deliver the selected doses of AVP. The mini osmotic pumps remained implanted until sacrifice. Physiological parameters (weight, systolic and diastolic blood pressure) were measured at GD 3, 13, 14, 18, and 20 using the MRBP tail-cuff BP monitor (IITC Life Sciences Inc., USA). Urine samples (24 h) were collected on GD 10, 17, and 21 to measure urinary proteins, using the M-TP Microprotein Kit (Beckman Coulter, CA, USA) and read using the SYNCHRON LX® System (Beckman Coulter, CA, USA).

Experimental study

Twenty-four pregnant female Sprague-Dawley rats (200–220 g) were randomly grouped as follows:

• Group 1: (n = 6) Non-pregnant with saline delivery (NS)

• Group 2: (n = 6) Non-pregnant with AVP delivery (NAVP)

• Group 3: (n = 6) Pregnant with saline delivery (PS)

• Group 4: (n = 6) Pregnant with AVP delivery (PAVP)

Based on the pilot study outcomes, an AVP dose of 150 ng/h was chosen since it produced the most significant changes in blood pressure and proteinuria compared to the lower doses tested. The ALZET mini osmotic pumps were subcutaneously implanted on GD 1 and remained implanted until sacrifice (GD 18) in all study groups. Saline and AVP (150 ng/h) were subcutaneously delivered throughout gestation, to the control and experimental groups respectively and pumps were removed at sacrifice (GD 18). Systolic and diastolic blood pressure was measured on GD 8, 14, and 18 using the MRBP tail-cuff BP monitor (IITC Life Sciences Inc., USA), by placing animals in a suitably sized restrainer. Normal blood pressure was defined as systolic ≤120 mmHg and diastolic ≤80 mmHg. Hypertension in rats was defined as systolic ≥140 mmHg and diastolic ≥90 mmHg (Brown et al. 2018). Animals were housed in metabolic cages (Techniplast, Italy) on GD 8, 14, and 18, for the measurement of water intake and urinary output; and collection of 24 h urine samples. Urinary protein content was measured using the M-TP Microprotein Kit (Beckman Coulter, CA, USA) and read using the SYNCHRON LX® System (Beckman Coulter, CA, USA). Animals were then euthanized on GD 18, via isoflurane overdose (Safeline Pharmaceuticals, South Africa). Blood samples were collected via cardiac puncture and centrifuged for 15 min at 3500 rpm at 4°C. The number and weight of placentae and pups were recorded. Biochemical and hematological analysis was carried out by a pathology laboratory using rodent reference ranges.

Histological evaluation of the liver, kidney, and placenta

Liver, kidney, and placenta samples were fixed in 10% buffered formaldehyde and embedded into paraffin wax blocks as per standard laboratory procedure (Burton et al. 2014). Sections of liver, kidney, and placental tissue were cut (3 µm) using a rotary microtome and mounted onto frosted glass slides (ISOLAB GmbH Herstellung und Vertrieb von Laborgeräten, Eschau, Germany). Tissue sections were de-paraffinized and rehydrated for hematoxylin and eosin staining (Fischer et al. 2008). Stained sections were permanently mounted. Tissue sections were examined using the ZEISS Axio Imager 2 and images captured using Zen (Blue edition) software (Carl-Zeiss-Strasse, Oberkochen, Germany).

Statistical analysis

All statistical analyses were carried out using Stata (Version 12). All data were non-parametric and are summarized as median and interquartile range (IQR). The biochemical/hematological parameters in pregnant rats were compared with non-pregnant rats using the Mann–Whitney test. The Kruskal–Wallis and Dunn’s post hoc test was used to compare the medians between groups. A probability value of p < 0.05 was considered statistically significant.

Ethics approval for animal study

This study was approved by the Animal Research Ethics Committee, University of KwaZulu-Natal (UKZN) (AREC/046/017). All procedures were conducted as per the approved institutional standard protocols for animal research.

Authors' contributions

Conception and study design: NG, SB, BNM, SS. Conduction of experiments and data collection: SR, VP. Analysis and interpretation of the data: NG, SR, SB. Development and finalisation of the manuscript: NG, SR, TN, SB BNM, SS.

Acknowledgments

The authors are grateful for and acknowledge the Biomedical Resource Unit (BRU) for their use of the facilities and would like to thank the staff of the BRU for technical assistance with the animal study. The authors also wish to thank Prof. M. Santillan (Carver College of Medicine, University of Iowa, Iowa) and Prof. J. Grobe (Medical College of Wisconsin, Wisconsin) for their valuable input in the initial development of this study.

Disclosure statement

The author(s) report no conflict of interest.

Additional information

Funding

This work was supported by the National Research Foundation under Grant [number 107236 and 122014].