Isaac E. Stillman

Excess placental soluble fms-like tyrosine kinase 1 (sFlt1) may contribute to endothelial dysfunction, hypertension, and proteinuria in preeclampsia

Preeclampsia, a syndrome affecting 5% of pregnancies, causes substantial maternal and fetal morbidity and mortality. The pathophysiology of preeclampsia remains largely unknown. It has been hypothesized that placental ischemia is an early event, leading to placental production of a soluble factor or factors that cause maternal endothelial dysfunction, resulting in the clinical findings of hypertension, proteinuria, and edema. Here, we confirm that placental soluble fms-like tyrosine kinase 1 (sFlt1), an antagonist of VEGF and placental growth factor (PlGF), is upregulated in preeclampsia, leading to increased systemic levels of sFlt1 that fall after delivery. We demonstrate that increased circulating sFlt1 in patients with preeclampsia is associated with decreased circulating levels of free VEGF and PlGF, resulting in endothelial dysfunction in vitro that can be rescued by exogenous VEGF and PlGF. Additionally, VEGF and PlGF cause microvascular relaxation of rat renal arterioles in vitro that is blocked by sFlt1. Finally, administration of sFlt1 to pregnant rats induces hypertension, proteinuria, and glomerular endotheliosis, the classic lesion of preeclampsia. These observations suggest that excess circulating sFlt1 contributes to the pathogenesis of preeclampsia.

Soluble endoglin contributes to the pathogenesis of preeclampsia.

Preeclampsia is a pregnancy-specific hypertensive syndrome that causes substantial maternal and fetal morbidity and mortality. Maternal endothelial dysfunction mediated by excess placenta-derived soluble VEGF receptor 1 (sVEGFR1 or sFlt1) is emerging as a prominent component in disease pathogenesis. We report a novel placenta-derived soluble TGF-β coreceptor, endoglin (sEng), which is elevated in the sera of preeclamptic individuals, correlates with disease severity and falls after delivery. sEng inhibits formation of capillary tubes in vitro and induces vascular permeability and hypertension in vivo. Its effects in pregnant rats are amplified by coadministration of sFlt1, leading to severe preeclampsia including the HELLP (hemolysis, elevated liver enzymes, low platelets) syndrome and restriction of fetal growth. sEng impairs binding of TGF-β1 to its receptors and downstream signaling including effects on activation of eNOS and vasodilation, suggesting that sEng leads to dysregulated TGF-β signaling in the vasculature. Our results suggest that sEng may act in concert with sFlt1 to induce severe preeclampsia.

Expanded double negative T cells in patients with systemic lupus erythematosus produce IL-17 and infiltrate the kidneys.

Double negative (DN) T cells are expanded in patients with systemic lupus erythematosus (SLE) and stimulate autoantibody production as efficiently as CD4+ T cells. In this study, we demonstrate that DN T cells from patients with SLE produce significant amounts of IL-17 and IFN-γ, and expand when stimulated in vitro with an anti-CD3 Ab in the presence of accessory cells. Furthermore, IL-17+ and DN T cells are found in kidney biopsies of patients with lupus nephritis. Our findings establish that DN T cells produce the inflammatory cytokines IL-17 and IFN-γ, and suggest that they contribute to the pathogenesis of kidney damage in patients with SLE.

Adenoviral gene therapy leads to rapid induction of multiple chemokines and acute neutrophil-dependent hepatic injury in vivo.

Replication-deficient adenoviruses are known to induce acute injury and inflammation of infected tissues, thus limiting their use for human gene therapy. However, molecular mechanisms triggering this response have not been fully defined. To characterize this response, chemokine expression was evaluated in DBA/2 mice following the intravenous administration of various adenoviral vectors. Administration of adCMVbeta gal, adCMV-GFP, or FG140 intravenously rapidly induced a consistent pattern of C-X-C and C-C chemokine expression in mouse liver in a dose-dependent fashion. One hour following infection with 10(10) PFU of adCMVbeta gal, hepatic levels of MIP-2 mRNA were increased >60-fold over baseline. MCP-1 and IP-10 mRNA levels were also increased immediately following infection with various adenoviral vectors, peaking at 6 hr with >25- and >100-fold expression, respectively. Early induction of RANTES and MIP-1beta mRNA by adenoviral vectors also occurred, but to a lesser degree. The induction of chemokines occurred independently of viral gene expression since psoralen-inactivated adenoviral particles produced an identical pattern of chemokine gene transcription within the first 16 hr of administration. The expression of chemokines correlated as expected with the influx of neutrophils and CD11b+ cells into the livers of infected animals. At high titers, all adenoviral vectors caused significant hepatic necrosis and apoptosis following systemic administration to DBA/2 mice. To investigate the role of neutrophils in this adenovirus-induced hepatic injury, animals were pretreated with neutralizing anti-MIP-2 antibodies or depleted of neutrophils. MIP-2 antagonism and neutrophil depletion both resulted in reduced serum ALT/AST levels and attenuation of the adenovirus-induced hepatic injury histologically, confirming that this early injury is largely due to chemokine production and neutrophil recruitment. Our findings further clarify the early immune response against replication-deficient adenoviral vectors and suggest a strategy to prevent adenovirus-mediated inflammation and tissue injury by interfering with chemokine or neutrophil function.

Activated vitamin D attenuates left ventricular abnormalities induced by dietary sodium in Dahl salt-sensitive animals

Observations in hemodialysis patients suggest a survival advantage associated with activated vitamin D therapy. Left ventricular (LV) structural and functional abnormalities are strongly linked with hemodialysis mortality. Here, we investigated whether paricalcitol (PC, 19-nor-1,25(OH)2D2), an activated vitamin D compound, attenuates the development of LV abnormalities in the Dahl salt-sensitive (DSS) rat and whether humans demonstrate comparable findings. Compared with DSS rats fed a high-salt (HS) diet (6% NaCl for 6 weeks), HS+PC was associated with lower heart and lung weights, reduced LV mass, posterior wall thickness and end diastolic pressures, and increased fractional shortening. Blood pressures did not significantly differ between the HS groups. Plasma brain natriuretic peptide levels, and cardiac mRNA expression of brain natriuretic peptide, atrial natriuretic factor, and renin were significantly reduced in the HS+PC animals. Microarray analyses revealed 45 specific HS genes modified by PC. In a retrospective pilot study of hemodialysis patients, PC-treated subjects demonstrated improved diastolic function and a reduction in LV septal and posterior wall thickness by echocardiography compared with untreated patients. In summary, PC attenuates the development of LV alterations in DSS rats, and these effects should be examined in human clinical trials.

PGC-1α promotes recovery after acute kidney injury during systemic inflammation in mice.

Sepsis-associated acute kidney injury (AKI) is a common and morbid condition that is distinguishable from typical ischemic renal injury by its paucity of tubular cell death. The mechanisms underlying renal dysfunction in individuals with sepsis-associated AKI are therefore less clear. Here we have shown that endotoxemia reduces oxygen delivery to the kidney, without changing tissue oxygen levels, suggesting reduced oxygen consumption by the kidney cells. Tubular mitochondria were swollen, and their function was impaired. Expression profiling showed that oxidative phosphorylation genes were selectively suppressed during sepsis-associated AKI and reactivated when global function was normalized. PPARγ coactivator-1α (PGC-1α), a major regulator of mitochondrial biogenesis and metabolism, not only followed this pattern but was proportionally suppressed with the degree of renal impairment. Furthermore, tubular cells had reduced PGC-1α expression and oxygen consumption in response to TNF-α; however, excess PGC-1α reversed the latter effect. Both global and tubule-specific PGC-1α-knockout mice had normal basal renal function but suffered persistent injury following endotoxemia. Our results demonstrate what we believe to be a novel mechanism for sepsis-associated AKI and suggest that PGC-1α induction may be necessary for recovery from this disorder, identifying a potential new target for future therapeutic studies.

Mitochondrial-targeted antioxidants represent a promising approach for prevention of cisplatin-induced nephropathy.

Cisplatin is a widely used antineoplastic agent; however, its major limitation is the development of dose-dependent nephrotoxicity whose precise mechanisms are poorly understood. Here we show not only that mitochondrial dysfunction is a feature of cisplatin nephrotoxicity, but also that targeted delivery of superoxide dismutase mimetics to mitochondria largely prevents the renal effects of cisplatin. Cisplatin induced renal oxidative stress, deterioration of mitochondrial structure and function, an intense inflammatory response, histopathological injury, and renal dysfunction. A single systemic dose of mitochondrially targeted antioxidants, MitoQ or Mito-CP, dose-dependently prevented cisplatin-induced renal dysfunction. Mito-CP also prevented mitochondrial injury and dysfunction, renal inflammation, and tubular injury and apoptosis. Despite being broadly renoprotective against cisplatin, Mito-CP did not diminish cisplatins antineoplastic effect in a human bladder cancer cell line. Our results highlight the central role of mitochondrially generated oxidants in the pathogenesis of cisplatin nephrotoxicity. Because similar compounds seem to be safe in humans, mitochondrially targeted antioxidants may represent a novel therapeutic approach against cisplatin nephrotoxicity.

Transcriptionally Active Syncytial Aggregates in the Maternal Circulation May Contribute to Circulating Soluble Fms-Like Tyrosine Kinase 1 in Preeclampsia

The cardinal manifestations of the pregnancy-specific disorder preeclampsia, new-onset hypertension, and proteinuria that resolve with placental delivery have been linked to an extracellular protein made by the placenta, soluble fms-like tyrosine kinase 1 (sFlt1), that injures the maternal vasculature. However, the mechanisms by which sFlt1, which is heavily matrix bound, gain access to the systemic circulation remain unclear. Here we report that the preeclamptic placentas outermost layer, the syncytiotrophoblast, forms abundant “knots” that are enriched with sFlt1 protein. These syncytial knots easily detach from the syncytiotrophoblast, resulting in free, multinucleated aggregates (50–150 &mgr;m diameter) that are loaded with sFlt1 protein and mRNA, are metabolically active, and are capable of de novo gene transcription and translation. At least 25% of the measurable sFlt1 in the third-trimester maternal plasma is bound to circulating placental microparticles. We conclude that detachment of syncytial knots from the placenta results in free, transcriptionally active syncytial aggregates that represent an autonomous source of sFlt1 delivery into the maternal circulation. The process of syncytial knot formation, shedding of syncytial aggregates, and appearance of placental microparticles in the maternal circulation appears to be greatly accelerated in preeclampsia and may contribute to the maternal vascular injury that characterizes this disorder.

Acute Tubular Necrosis Is a Syndrome of Physiologic and Pathologic Dissociation

Acute tubular necrosis (ATN) is a syndrome of intrinsic renal failure secondary to ischemic or toxic insults. The histopathologic findings of ATN are inconstant. When present, they are limited to the tubulo-interstitium and often subtle despite profound dysfunction. Experimental models of ATN in healthy animals commonly use single insults that result in extensive injury, circumstances that do not parallel the human situation. Recently, there has been a shift to more clinically relevant models using an acute insult superimposed on predisposing factors. This review discusses the complex hemodynamic interrelationships of hypoxia, tubular injury, and altered glomerular filtration, suggesting new ways to understand the pathophysiology of ATN.

On the Intraoperative Molecular Status of Renal Allografts after Vascular Reperfusion and Clinical Outcomes

Many hypothesize that subtle inflammation and immune activity detected in the intraoperative period are linked to adverse postkidney transplant clinical outcomes. To this end, renal allografts were analyzed for expression of pro-inflammatory, inflammation-induced adhesion molecules, immune activation as well as anti-apoptotic genes expressed 15 min after vascular reperfusion (zero-hour) to determine whether this analysis can aid in predicting the occurrence of delayed graft function (DGF), acute rejection (AR), and the quality of graft function at 6 mo. Intraoperative biopsies were obtained from 75 consecutively performed renal allografts in which consent was obtained 15 min after vascular reperfusion. These biopsies were analyzed by quantitative real-time PCR for transcription of 15 select genes and by standard histopathology. Posttransplant clinical outcomes were also analyzed in respect to intraoperative transcriptional profiles and clinical parameters available at the time of transplantation. This study demonstrates that a limited and hypothesis-driven PCR-based transcriptional profile of the zero-hour kidney biopsy predicts posttransplant clinical outcomes including DGF, early AR, and the quality of renal function 6 mo posttransplantation. For some clinical endpoints, the combined use of molecular analysis and established clinical indicators available at the time of transplantation further enhances the quality of prognosis. The transcriptional profiling data provide absolutely essential data to the predictive models, particularly with respect to AR and renal function 6 mo posttransplantation.