Kidney Injury Caused by Preeclamptic Pregnancy Recovers Postpartum in a Transgenic Rat Model

Abstract

Preeclampsia (PE) is characterized by the onset of hypertension (≥140/90 mmHg) and presence of proteinuria (>300 mg/L/24 h urine) or other maternal organ dysfunctions. During human PE, renal injuries have been observed. Some studies suggest that women with PE diagnosis have an increased risk to develop renal diseases later in life. However, in human studies PE as a single cause of this development cannot be investigated. Here, we aimed to investigate the effect of PE on postpartum renal damage in an established transgenic PE rat model. Female rats harboring the human-angiotensinogen gene develop a preeclamptic phenotype after mating with male rats harboring the human-renin gene, but are normotensive before and after pregnancy. During pregnancy PE rats developed mild tubular and glomerular changes assessed by histologic analysis, increased gene expression of renal damage markers such as kidney injury marker 1 and connective-tissue growth factor, and albuminuria compared to female wild-type rats (WT). However, four weeks postpartum, most PE-related renal pathologies were absent, including albuminuria and elevated biomarker expression. Only mild enlargement of the glomerular tuft could be detected. Overall, the glomerular and tubular function were affected during pregnancy in the transgenic PE rat. However, almost all these pathologies observed during PE recovered postpartum.

Keywords: kidney injury; postpartum; preeclampsia; transgenic rat model.

Figure 1

Hypertension developed during pregnancy in a preeclamptic rat model. (A) Female Sprague–Dawley rats transgenic for the human angiotensinogen gene (Tg(Agt)) were mated with male Sprague–Dawley rats transgenic for the human renin gene (Tg(Ren)). Thereafter, female rats developed a preeclamptic phenotype (PE). Sprague–Dawley wild-type rats (WT) were mated, and females served as a control. (B) Exemplary illustration of the course of the mean arterial blood pressure is shown for PE and WT over a period of 49 days, including pregnancy. It rose from day 12 to a maximum of 170 mmHg and recurred shortly after pregnancy in preeclamptic dams. Control rats did not develop hypertension. All values are presented as mean ± SD (n = 5–9). Timepoints of metabolic cage and sacrifice are indicated. (C) PE rats developed hypertension during pregnancy, mean arterial blood pressure was significantly increased (day 19) compared to WT and recovered postpartum (day 49). (D) Albuminuria was detected as elevated urine albumin to creatinine ratio (n = 8) in preeclamptic rats during (day 19) and after pregnancy (day 49) compared to pregnant WT. A statistical analysis was not conducted for the course of blood pressure. Statistical analysis was performed using a two-way ANOVA. Bars are shown as mean ± SD (n = 8–11). p-value is shown as ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.001 (between groups).

Figure 2

Renal injury marker expression was altered during and slightly after preeclamptic pregnancy. (A) Descriptive overview of hypothesis-driven relative gene expression in kidney tissue during preeclamptic (PE) and control (WT) pregnancy (day 21) and postpartum (day 50). Analyzed genes are marker for nephron injury, angiogenesis, inflammation, fibrosis, and oxidative stress (from top to bottom). Legend indicates means of relative gene expression normalized to 18S expression. (B–F) Marker genes that show altered expression in the PE group are emphasized. (B,C) Nephron injury marker kidney injury molecule-1 (Kim-1) and neutrophil gelatinase-associated lipocalin 2 (Ngal) were elevated during preeclamptic pregnancy compared to WT. (D) Fibrosis marker connective tissue growth factor (Ctgf) expression was slightly increased during preeclamptic pregnancy. (E,F) Oxidative stress marker expression of neutrophil cytosolic factor 4 (Ncf-4) and NADPH oxidase 4 (Nox-4) was higher during and/or after preeclamptic pregnancy. All genes are normalized to 18s expression. Statistical analysis was performed using a two-way ANOVA. Bars are indicated as mean ± SD (n = 5–7). p-value is shown in comparison to respective controls as * p ≤ 0.05; *** p ≤ 0.001.

Figure 3

Minimal renal alterations during preeclamptic rat pregnancy. (A) Quantification of glomerular tuft area (asterisk) in kidneys of preeclampsia (PE) and control (WT) rats showed no glomerular enlargement during but after preeclamptic pregnancy. Representative images of hemalaun–eosin-stained glomeruli in kidney sections of PE and WT rats are shown. (B) The quantification of mean periodic acid–Schiff (PAS)-positive glomerular area displayed a significantly higher density in PE at day 21. Mild glomerular sclerosis was visible in the respective images of PAS-stained glomeruli. (C) Semi-quantitative tubular injury analysis revealed no significant difference between groups, though a slight tendency to increased damage was visible in PE at day 21. Total tubular injury score was gathered in a blinded manner in percent. Exemplary images of hemalaun–eosin-stained proximal tubules displayed mild tubular dilation and brush border thinning (arrows) at PE day 21. (D) Inflammation was not significantly higher in preeclamptic kidney tissue. CD68-positive cells were counted in ten fields of view per kidney. Representative images of immunohistochemically stained kidney sections for WT and PE were counterstained with DAPI (blue). Primary mouse anti-rat CD68 antibody (clone ED1) and secondary Cy3-conjugated anti-mouse antibody (red) were used. (E) PE did not cause perivascular fibrosis in kidney tissue. Relative fibrotic vessel area (arrow head) did not show differences between preeclamptic (PE) and control (WT) rats at all time points. Example images of Masson–Goldner trichrome-stained kidney sections displayed similar-sized connective tissue areas of the arteries. Bars are shown as mean ± SD (n = 5), analyzed with a two-way ANOVA. p-values are indicated as * p ≤ 0.05 (between groups). Respective images are presented with scale bars (40 µm).

Figure 4

Urinary parameters were altered during PE and mostly restored after pregnancy. Amounts of urine creatinine (A), sodium (B), potassium (C), and chloride (D) are displayed per day. All parameters were reduced on day 19 of preeclamptic pregnancy (PE) compared to respective controls (WT). Urine creatinine was less reduced in PE on day 49. Sodium, potassium, and chloride recovered after PE pregnancy. Statistical analysis was done with a two-way ANOVA. Mean ± SD is indicated (n = 7–8). p-value is shown * p ≤ 0.05; ** p ≤ 0.01; **** p ≤ 0.0001.