Objective-The role of hypercoagulability in the pathogenesis of diabetic nephropathy (DN) remains elusive. We recently reported the increased infiltration of macrophages expressing tissue factor in diabetic kidney glomeruli; tissue factor activates coagulation factor X (FX) to FXa, which in turn stimulates protease-activated receptor 2 (PAR2) and causes inflammation. Approach and Results-Here, we demonstrated that diabetes mellitus increased renal FX mRNA, urinary FXa activity, and FX expression in glomerular macrophages. Administration of an oral FXa inhibitor, edoxaban, ameliorated DN with concomitant reductions in the expression of PARs (Par1 and Par2) and of proinflammatory and profibrotic genes. Diabetes mellitus induced PAR2, and lack of Par2 ameliorated DN. FXa or PAR2 agonist increased inflammatory cytokines in endothelial cells and podocytes in vitro. Conclusions-We conclude that enhanced FXa and PAR2 exacerbate DN and that both are promising targets for preventing DN. Alleviating inflammation is probably more important than inhibiting coagulation per se when treating kidney diseases using anticoagulants.

Preeclampsia (PE) is a pregnancy-induced hypertension with proteinuria that typically develops after 20 weeks of gestation. A reduction in uterine blood flow causes placental ischemia and placental release of anti-angiogenic factors such as sFlt-1 followed by PE. Although the reduced uterine perfusion pressure (RUPP) model is widely used in rats, investigating the role of genes on PE using genetically engineered animals has been problematic because it has been difficult to make a useful RUPP model in mice. To establish a RUPP model of PE in mice, we bilaterally ligated ovarian vessels distal to ovarian branches, uterine vessels, or both in ICR-strain mice at 14.5 days post coitum (dpc). Consequently, these mice had elevated BP, increased urinary albumin excretion, severe endotheliosis, and mesangial expansion. They also had an increased incidence of miscarriage and premature delivery. Embryonic weight at 18.5 dpc was significantly lower than that in sham mice. The closer to the ligation site the embryos were, the higher the resorption rate and the lower the embryonic weight. The phenotype was more severe in the order of ligation at the ovarian vessels < uterine vessels < both. Unlike the RUPP models described in the literature, this model did not constrict the abdominal aorta, which allowed BP to be measured with a tail cuff. This novel RUPP model in mice should be useful for investigating the pathogenesis of PE in genetically engineered mice and for evaluating new therapies for PE.

Preeclampsia (PE) complicates ∼5% of human pregnancies and is one of the leading causes of pregnancy-related maternal deaths. The only definitive treatment, induced delivery, invariably results in prematurity, and in severe early-onset cases may lead to fetal death. Many currently available antihypertensive drugs are teratogenic and therefore precluded from use. Nonteratogenic antihypertensives help control maternal blood pressure in PE, but results in preventing preterm delivery and correcting fetal growth restriction (FGR) that also occurs in PE have been disappointing. Here we show that dietary nicotinamide, a nonteratogenic amide of vitamin B 3 , improves the maternal condition, prolongs pregnancies, and prevents FGR in two contrasting mouse models of PE. The first is caused by endotheliosis due to excess levels in the mothers of a soluble form of the receptor for vascular endothelial growth factor (VEGF), which binds to and inactivates VEGF. The second is caused by genetic absence of Ankiryn-repeat-and-SOCS-box-containingprotein 4, a factor that contributes to the differentiation of trophoblast stem cells into the giant trophoblast cells necessary for embryo implantation in mice; its absence leads to impaired placental development. In both models, fetal production of ATP is impaired and FGR is observed. We show here that nicotinamide decreases blood pressure and endotheliosis in the mothers, probably by inhibiting ADP ribosyl cyclase (ADPRC), and prevents FGR, probably by normalizing fetal ATP synthesis via the nucleotide salvage pathway. Because nicotinamide benefits both dams and pups, it merits evaluation for preventing or treating PE in humans.he maternal hypertension and proteinuria characterizing preeclampsia (PE) are primarily consequences of an imbalance between proangiogenic growth factors that promote vascular well-being (such as VEGF), and antiangiogenic factors that sequester the growth factors (such as the soluble form of VEGF receptor-1, now referred to as sFLT1) (1). Both the hypertension and the proteinuria of PE are caused by abnormally high amounts of antiangiogenic factors derived from the placenta. Fetal growth restriction (FGR), an additional feature of PE, is a consequence of reduced placental blood flow resulting from damage to the placental vasculature caused by antiangiogenic factors and/or to impaired development of the placenta. Endothelin-1 (EDN1) is the most powerful naturally occurring prohypertensive peptide, and antagonists of the endothelin type A receptor (EDNRA) greatly ameliorate the PE-like condition that develops in the kidneys of rodents with excess sFLT1 (2, 3). Unfortunately, these antagonists are teratogenic (4) and consequently unacceptable for use in treating PE.Nicotinamide is a potential nonteratogenic alternative because it relaxes blood vessels constricted with EDN1 (5) and because it has been extensively tested at high oral doses in men and (nonpregnant) women and found safe (6). Results and DiscussionNicotinamide Ameliorates the Hypertension, Albuminuria, an...

Liver dysfunction is a major problem in patients with severe preeclampsia (PE), hemolysis, elevated liver enzymes, and low platelet count (HELLP) syndrome, or in patients receiving anti-vascular endothelial growth factor (VEGF) therapy. Excessive soluble fms-like tyrosine kinase 1 (sFlt1) that antagonizes VEGF has been implicated in the pathogenesis of PE. VEGF increases the expression of endothelial nitric oxide synthase (eNOS) and activates it. eNOS polymorphisms that cause reduced NO production are associated with PE. The aim of this study was to clarify the role on hepatic function by excess sFlt1 in the absence of eNOS gene product. We first overexpressed sFlt1 using adenovirus in eNOS −/− and eNOS +/+ mice. Excessive sFlt1 and lack of eNOS synergistically increased plasma levels of liver transaminases, exacerbated infiltration of inflammatory cells, elevated expression levels of cytokines in the liver, and aggravated oxidative stress and coagulation abnormalities. Lack of eNOS in the presence of excess sFlt1 also induced thrombocytopenia, whereas eNOS +/+ mice with excess sFlt1 alone showed no or modest liver phenotype. Taken together, excessive sFlt1 and lack of eNOS synergistically induce hepatic dysfunction and thrombocytopenia, suggesting a novel role for VEGF and nitric oxide signaling in hepatocyte-endothelial cross-talk in health and in liver injury states.

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Fetal growth restriction (FGR) is a risk factor exacerbating a poor neurological prognosis at birth. A disease exacerbating a poor neurological prognosis is cerebral palsy. One of the cause of this disease is cerebral hemorrhage including intraventricular hemorrhage. It is believed to be caused by an inability to autoregulate cerebral blood flow as well as immaturity of cerebral vessels. Therefore, if we can evaluate the function of autonomic nerve, cerebral hemorrhage risk can be predicted beforehand and appropriate delivery management may be possible. Here dysfunction of autonomic nerve in mouse FGR fetuses was evaluated and the relationship with cerebral hemorrhage incidence when applying hypoxic load to resemble the brain condition at the time of delivery was examined. Furthermore, FGR incidence on cerebral nerve development and differentiation was examined at the gene expression level. FGR model fetuses were prepared by ligating uterine arteries to reduce placental blood flow. To compare autonomic nerve function in FGR mice with that in control mice, fetal short term variability (STV) was measured from electrocardiograms. In the FGR group, a significant decrease in the STV was observed and dysfunction of cardiac autonomic control was confirmed. Among genes related to nerve development and differentiation, Ntrk and Neuregulin 1, which are necessary for neural differentiation and plasticity, were expressed at reduced levels in FGR fetuses. Under normal conditions, Neurogenin 1 and Neurogenin 2 are expressed mid-embryogenesis and are related to neural differentiation, but they are not expressed during late embryonic development. The expression of these two genes increased in FGR fetuses, suggesting that neural differentiation is delayed with FGR. Uterine and ovarian arteries were clipped and periodically opened to give a hypoxic load mimicking the time of labor, and the bleeding rate significantly increased in the FGR group. This suggests that FGR deteriorates cardiac autonomic control, which becomes a risk factor for cerebral hemorrhage onset at birth. This study demonstrated that cerebral hemorrhage risk may be evaluated before parturition for FGR management by evaluating the STV. Further, this study suggests that choosing an appropriate delivery timing and delivery method contributes to neurological prognosis improvement.

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Preeclampsia (PE) is pregnancy-induced hypertension with proteinuria that typically develops after 20 wk of gestation. Antihypertensives currently used for PE reduce blood pressure of PE mothers but do not prevent preterm delivery and do not alleviate fetal growth restriction (FGR) associated with PE. We have recently shown that the activation of the endothelin (ET) system exacerbates PE. However, ET receptor antagonists are teratogenic and not suitable for pregnant women. The vitamin B nicotinamide (Nam) inhibits vasoconstriction by ET and is generally considered safe and harmless to babies. Nam also alleviates oxidative stress, which exacerbates PE and FGR. The aim of the present study was to evaluate therapeutic effects of Nam on the PE-like phenotype using a reduced uterine perfusion pressure (RUPP) model in mice that we have recently developed. We bilaterally ligated uterine vessels of pregnant mice and administered Nam or water daily by gavage. Nam improved maternal hypertension, proteinuria, and glomerular endotheliosis in RUPP mice. Moreover, Nam prolonged pregnancies and improved survival and growth of the embryos in RUPP PE mice. In conclusion, Nam alleviates the PE-like phenotype and FGR in the murine RUPP model. Nam could help treat maternal hypertension and FGR in human PE.