Evidence of kidney injury in preeclampsia: Increased maternal and urinary levels of NGAL and KIM-1 and their enhanced expression in proximal tubule epithelial cells.

Background and objective: Proteinuria and glomerular endotheliosis are characteristics of glomerular injury in preeclampsia, a hypertensive disorder in human pregnancy. Neutrophil gelatinase-associated lipocalin (NGAL) and kidney injury molecule-1 (KIM-1) are biomarkers of acute/chronic renal tubule injury. To determine if tubule injury occurs in preeclampsia, we determined maternal plasma and urine NGAL and KIM-1 levels and evaluated NGAL and KIM-1 expression in kidney biopsy specimens from women with preeclampsia. Methods: Prenatal and postpartum maternal blood and urinary samples were collected from three groups of pregnant women: normal pregnancy (n = 100), preeclampsia (n = 83), and pregnancy complicated with chronic hypertension (n = 20). Plasma and urine levels of NGAL and KIM-1 were measured by ELISA. Kidney biopsy tissue sections from patients with preeclampsia (n = 5) were obtained from Pathology Archives and processed to determine NGAL and KIM-1 expression by immunostaining and high kidney solution images were assessed by electron microscopy (EM). Results: Prenatal plasma and urine levels of NGAL and KIM-1 were significantly higher in preeclamptic than in normal controls, p < 0.01. In normal pregnancy, both plasma and urine levels of NGAL and KIM-1 at 24-48 h after delivery and 6-8 weeks postpartum were relatively comparable to that of antenatal levels. In preeclampsia, urine, but not plasma, NGAL levels were reduced at 6-8 weeks postpartum compared to the antenatal levels, p < 0.05. Although maternal and urine KIM-1 levels were reduced at 6-8 weeks postpartum compared to the antenatal levels in preeclampsia, the levels were still higher than those in normal pregnancy. Positive expression of NGAL and KIM-1 was detected in proximal tubule epithelial cells in kidney tissue specimens from preeclampsia but not in non-pregnancy controls. EM examination showed glomerular and tubular injury in preeclampsia. Conclusion: Our findings of increased maternal levels and urine secretion of NGAL and KIM-1, along with the upregulation of NGAL and KIM-1 expression in tubular epithelial cells in preeclampsia, provide plausible evidence that tubular injury exists in preeclampsia. The higher postpartum NGAL and KIM-1 levels in preeclamptic pregnancies indicate that tubular injury would not resolve within 2-3 months after delivery and suggest that proper follow-up and management of kidney function in women with preeclampsia would be necessary to reduce chronic kidney diseases in those women later in life.

Cell-type specific distribution and activation of type I IFN pathway molecules at the placental maternal-fetal interface in response to COVID-19 infection.

Background and objective: COVID-19 infection in pregnancy significantly increases risks of adverse pregnancy outcomes. However, little is known how the innate immunity at the placental maternal-fetal interface responds to COVID-19 infection. Type I IFN cytokines are recognized as a key component of the innate immune response against viral infection. In this study, we specifically evaluated expression of IFN antiviral signaling molecules in placentas from women infected with COVID-19 during pregnancy. Methods: Expression of IFN activation signaling pathway molecules, including cyclic GMP-AMP synthase (cGAS), stimulator of interferon genes (STING), interferon regulatory factor 3 (IRF3), Toll-like receptor 7 (TLR7), mitochondrial antiviral-signaling protein (MAVS), and IFNß were determined in formalin-fixed paraffin embedded (FFPE) placental tissue sections (villous and fetal membrane) by immunostaining. A total of 20 placentas were examined, 12 from COVID-19 patients and 8 from non-COVID-19 controls. Patient demographics, clinical data, and placental pathology report were acquired via EPIC medical record review. Results: Except BMI and placental weight, there was no statistical difference between COVID and non-COVID groups in maternal age, gestational age at delivery, gravity/parity, delivery mode, and newborn gender and weight. In COVID-exposed group, the main pathological characteristics in the placental disc are maternal and fetal vascular malperfusion and chronic inflammation. Compared to non-COVID controls, expression of IFN activation pathway molecules were all upregulated with distinct cell-type specific distribution in COVID-exposed placentas: STING in villous and decidual stromal cells; IRF3 in cytotrophoblasts (CTs) and extra-villous trophoblasts (EVTs); and TLR7 and MAVS in syncytiotrophoblasts (STs), CTs, and EVTs. Upregulation of STING, MAVS and TLR7 was also seen in fetal endothelial cells. Conclusions: STING, IRF3, TLR7, and MAVS are key viral sensing molecules that regulate type I IFN production. Type I IFNs are potent antiviral cytokines to impair and eradicate viral replication in infected cells. The finding of cell-type specific distribution and activation of these innate antiviral molecules at the placental maternal-fetal interface provide plausible evidence that type I IFN pathway molecules may play critical roles against SARS-CoV-2 infection in the placenta. Our findings also suggest that placental maternal-fetal interface has a well-defined antiviral defense system to protect the developing fetus from SARS-CoV-2 infection.

Downregulation of miR-126-3p expression contributes to increased inflammatory response in placental trophoblasts in preeclampsia.

MiR-126-3p is a prototype of an endothelial miRNA and has protective effects on endothelial cells. However, little is known about the effects of miR-126-3p on placental trophoblasts. In the present study, we tested the hypothesis that aberrant miR-126-3p expression is present in preeclamptic placenta which contributes to increased inflammatory response in trophoblasts. Placentas were obtained immediately after delivery from normotensive and preeclamptic pregnancies. Villous tissue was either fixed with formalin or used for trophoblast isolation. Trophoblast miR-126-3p expression was assessed by in situ hybridization of formalin-fixed tissue sections and by RT-PCR in cultured syncytiotrophoblasts. Culture medium was collected for measurement of IL-6, TNFα, and 8-Isoprostane production by ELISA and total cellular protein was collected for evaluation of HIF1α expression by Western blot. Effects of overexpression of miR-126-3p in trophoblasts on cytokine production were tested by transfection of pre-mir-126, a precursor of miR-126, into primary isolated trophoblasts. We found that downregulation of miR-126-3p expression was associated with increased IL-6 and TNFα production in trophoblasts from preeclamptic placentas vs. normal placentas. Moreover, transient overexpression of miR-126-3p significantly reduced IL-6 and TNFα production in trophoblasts from both normal and preeclamptic placentas. We further found that increase in miR-126-3p expression not only suppressed hypoxia-induced increases in IL-6 and TNFα production, but also attenuated hypoxia-induced increases in HIF1α expression and 8-Isoprostane production in trophoblasts cultured under hypoxic condition. These results provide plausible evidence that downregulation of miR-126-3p expression reduces anti-inflammatory and anti-oxidative stress activities in placental trophoblasts in preeclampsia.

Upregulation of histone H3K9 methylation in fetal endothelial cells from preeclamptic pregnancies.

Adverse intrauterine environment has been considered a predisposing factor for fetal programming in preeclampsia. Using human umbilical vein endothelial cells (HUVECs), we specifically explored if aberrant histone methylation occurs in fetal endothelial cells in preeclampsia. Strikingly, we found that increased di-, and tri-methylation of histone H3 lysine 9 (H3K9me2 and H3K9me3) expression were associated with upregulation of methyltransferase G9a and downregulation of endothelial nitric oxide synthase and CuZn-SOD expression in preeclamptic HUVECs. We further demonstrated that hypoxia-induced hypermethylation of H3K9 and reduced CuZn-SOD expression mimicked what were seen in preeclamptic HUVECs and inhibition of G9a could attenuate these hypoxia-induced adverse events. Our study was the first to identify hypermethylation status in fetal endothelial cells in preeclampsia, which provides plausible evidence that increased oxidative stress in the intrauterine environment is likely a mechanism to induce aberrant histone modification in fetal endothelial cells which may have a significant impact on fetal programming in preeclampsia.

Maternal soluble PD-1 levels are significantly increased in women with preeclampsia.

PROBLEM: Programmed cell death-1 (PD-1) and its ligand (PD-L1) have emerged as key players in regulating immune tolerance. Preeclampsia is associated with maladaptation of immune tolerance during pregnancy. This study aimed to determine if maternal soluble PD-1 (sPD-1) and soluble PD-L1 (sPD-L1) levels are altered in preeclampsia. METHOD OF STUDY: Maternal sPD-1 and sPD-L1 levels were measured by ELISA in 172 pregnant women (86 normotensive and 86 preeclampsia). The differences in sPD-1 and sPD-L1 levels between normotensive and preeclamptic pregnant women, <34 vs >34 weeks, and fetal gender differences were assessed. Data were analyzed by unpaired t test or chi-square. A probability level of <.05 was considered statistically significant. RESULTS: Maternal sPD-1 levels were significantly higher in preeclamptic than in normotensive pregnant women, 6262 ± 1860 vs 1134 ± 349 pg/mL, P < .01. sPD-1 levels were not statistically different between <34 and >34 weeks of gestation in both normotensive and preeclamptic groups. sPD-1 levels were relatively higher in mothers with female fetus than with male fetus in the preeclamptic group: 8104 ± 3054 vs 3802 ± 2177 pg/mL, but relatively lower in mothers with female fetus than with male fetus in the normotensive group: 425 ± 134 vs 625 ± 182 pg/mL. Maternal sPD-L1 levels were relatively higher in preeclamptic than in normotensive pregnant women: 143 ± 52 vs 69 ± 13 pg/mL. CONCLUSION: Aberrant sPD-1/sPD-L1 signaling is present in preeclampsia. Whether increased maternal sPD-1 and sPD-L1 levels were associated with fetal gender difference or immune tolerance dissimilarity during pregnancy in women with preeclampsia warrants further investigation.

Downregulation of vitamin D receptor and miR-126-3p expression contributes to increased endothelial inflammatory response in preeclampsia.

PROBLEM: To investigate whether downregulation of miR-126-3p and vitamin D receptor (VDR) expression contributes to increased endothelial inflammatory response in preeclampsia. METHODS OF STUDY: Maternal vessel miR-126-3p expression was assessed by in situ hybridization. VDR expression and VCAM-1 expression were determined by immunostaining. Subcutaneous adipose tissue sections from normotensive and preeclamptic pregnant women were used. HUVECs from normotensive deliveries were used to test anti-inflammatory effects of vitamin D and miR-126-3p in endothelial cells (ECs) treated with TNFα in vitro. 1,25(OH)2 D3 was used as bioactive vitamin D. Transient overexpression of miR-126-3p in ECs was induced by transfection of pre-mir-126 precursor. Endothelial VCAM-1 and SOCS-3 expression or production was determined by Western blotting or by ELISA, respectively. RESULTS: Reduced VDR and miR-126-3p expression, but increased VCAM-1 expression, was observed in maternal vessel endothelium in tissue sections from women with preeclampsia compared to normotensive pregnant controls. Transient overexpression of miR-126-3p not only attenuated upregulation of VCAM-1 expression and production, but also preserved downregulation of SOCS-3 expression, induced by TNFα in ECs. VDR expression and miR-126-3p expression were significantly upregulated in cells treated with 1,25(OH)2 D3 , but not in cells transfected with VDR siRNA. CONCLUSION: Downregulation of VDR and miR-126-3p expression was associated with upregulation of VCAM-1 expression in systemic vessel endothelium in preeclampsia. The finding of increased anti-inflammatory property by 1,25(OH)2 D3 through promotion of VDR and miR-126-3p expression in ECs provide plausible evidence that vitamin D deficiency and downregulation of VDR expression could contribute to increased inflammatory phenotypic changes in maternal vasculature in preeclampsia.

Aberrant pro-atrial natriuretic peptide/corin/natriuretic peptide receptor signaling is present in maternal vascular endothelium in preeclampsia.

OBJECTIVE: Corin is a serine protease that converts pro-atrial natriuretic peptide (pro-ANP) to atrial natriuretic peptide (ANP), a cardiac hormone that regulates salt-water balance and blood pressure. ANP is degraded by natriuretic peptide receptor (NPR). This study was to determine if aberrant pro-ANP/corin/NPR signaling is present in maternal vascular system in preeclampsia. STUDY DESIGN: Maternal venous blood was obtained from 197 pregnant women (84 normotensive, 16 complicated with chronic hypertension (CHT), 11 mild and 86 severe preeclampsia). Plasma corin and pro-ANP concentrations were measured by enzyme-linked immunosorbent assay. Maternal subcutaneous fat tissue was obtained from 12 pregnant women with cesarean section delivery (6 normotensive and 6 preeclampsia). Vascular ANP and its receptors NPR-A, NPR-B, and NPR-C expression were examined by immunostaining of paraffin embedded subcutaneous fat tissue sections. RESULTS: Corin concentrations were significantly higher in mild (2.78 ±â€¯0.67 ng/ml, p < .05) and severe (2.53 ±â€¯0.18 ng/ml, p < .01) preeclampsia than in normotensive (1.58 ±â€¯0.08 ng/ml) and CHT (1.55 ±â€¯0.20 ng/ml) groups. Pro-ANP concentrations were significantly higher in CHT (1.59 ±â€¯0.53 ng/ml, p < .05) and severe preeclampsia (1.42 ±â€¯0.24 ng/ml, p < .01) than in normotensive (0.48 ±â€¯0.06 ng/ml) and mild preeclampsia (0.52 ±â€¯0.09 ng/ml) groups. ANP and NPR-B expression was undetectable in maternal vessels from normotensive and preeclamptic pregnancies, but reduced NPR-A expression and increased NPR-C expression was found in maternal vessel endothelium in preeclampsia. CONCLUSIONS: ANP is a vasodilator and NPR-C is a clearance receptor for ANP. The finding of upregulation of NPR-C expression suggests that circulating ANP clearance or degradation is increased in preeclampsia. These results also suggest that pro-ANP/corin/NPR signaling is dominant in the vascular system in preeclampsia.