Expression of NEDD4L and ENaC in Urinary Extracellular Vesicles in Pre-eclampsia

Figures & data

Table 1. Primary antibodies.

Antibody target	Serial number	Antibody type	Company
NEDD4L	N/A	Rabbit-derived polyclonal antibody	In-house antibody (19)
α-ENaC	SPC-403	Rat-derived polyclonal antibody against N-terminal amino acid 46–68	StressMarq Biosciences Inc., Canada
γ-ENaC	SPC-405	Rat-derived polyclonal antibody against C terminal amino acid 629–650	StressMarq Biosciences Inc., Canada
Tumour susceptibility gene 101 (TSG101)	T5701	Rabbit-derived polyclonal antibody	Sigma-AldrichMissouri, USA Missouri, USA
CD9	D801A	Rabbit-derived monoclonal antibody	Cell Signaling TechnologyMassachusetts, USA Massachusetts, USA

Table 2. Characteristics of subjects in the normotensive pregnant and pre-eclamptic groups.

	NP (n = 17)	PE (n = 19)	p
Age (years)	30.1 ± 4.0	31.7 ± 5.2	.33
Gestation (weeks)	37.1 ± 2.7	30.3 ± 3.8	<.0001
Systolic blood pressure (mmHg)	115 ± 9	155 ± 17	<.0001
Diastolic blood pressure (mmHg)	71 ± 8	94 ± 12	<.0001
Urine protein/creatinine ratio (mg/mmol)	N/A1	135 (72–528)
Urine creatinine (mmol/L)	7.8 ± 6.1	7.7 ± 5.4	.89
Body mass index (kg/m^2)	27 ± 6.9	33 ± 11.0	.06
Number of babies with birthweight below 10th percentile adjusted for gestational age^2	0	9 (47%)	<.01
Serum biochemistry^3
Sodium (mmol/L)	138.2 ± 1.9	135.7 ± 3.4	.03
Potassium (mmol/L)	4.1 ± .3	4.6 2 ± .5	<.01
Bicarbonate (mmol/L)	22.3 ± 2.83	2.81 ± 2.4	.13
Creatinine (µmol/L)	53.0 ± 8.8	59.7 ± 11.6	.10

NP, normotensive pregnant group; PE, pre-eclamptic group. Data expressed as mean ± standard deviation, except for urine protein/creatinine ratio which is expressed as median (interquartile range).

¹Urine protein/creatinine ratio was not measured in the normotensive pregnant group.

²Percentile of birthweight adjusted for gestational age was determined according to the reference range proposed by Joseph et al. (29). Birthweight data was missing for four normotensive pregnant patients.

³In the NP group, serum biochemistry was not performed in five subjects and therefore 12 subjects were included in the analyses. Normal ranges of serum biochemistry for sodium 135-145 mmol/L, potassium 3.5–5.2 mmol/L, and bicarbonate 22-32 mmol/L.

Figure 1. Expression of antibodies directed against NEDD4L, α-ENaC and γ-ENaC on Western blot in normotensive pregnant controls (NP, n = 17) and pre-eclamptic patients (PE, n = 19) normalized to spot urine creatinine (mmol/L). Data are presented as scatter plots, with each patient’s densitometry value represented as a dot and the horizontal line is the median of the sample. (a) A representative Western blot of four NP patients and four PE patients with antibodies against NEDD4L, α-ENaC, and γ-ENaC, as well as vesicle markers TSG101 and CD9. (b) Densitometry analysis of NEDD4L (110 and 130kDa bands) showed no difference between the two groups (p = 0.17). (c) The 30kDa band of α-ENaC was quantified. Densitometry analysis did not show a significantly increased expression of α-ENaC in PE group compared to NP (p = 0.10). (d) For γ-ENaC, the 70kDa band was quantified. Densitometry analysis showed a 6.9-fold increased expression of γ-ENaC in PE group compared to NP (p < 0.0001). (e) Percentage of α-ENaC expression in NP and PE groups. (f) Percentage of γ-ENaC expression in NP and PE groups.

Figure 2. Regression analyses of NEDD4L with detectable α-ENaC and γ-ENaC in normotensive pregnant controls (NP) and pre-eclamptic patients (PE). (a) In the normotensive pregnant group, there were no significant correlations between NEDD4L and α-ENaC (p = 0.97), as well as NEDD4L and γ-ENaC (p = 0.12). (b) In the pre-eclampsia group, there was a significant correlation between Nedd4-2 and γ-ENaC (p = 0.01) but not with NEDD4L and α-ENaC (p = 0.35). Patients without detectable ENaC were not included in the regression analysis.

Joseph FA, Hyett JA, Schluter PJ, et al. New Australian birthweight centiles. Med J Aust. 2020 Jul;213(2):79–85.

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