Tatiana Novitskaya

Histone deacetylase inhibitor enhances recovery after AKI.

At present, there are no effective therapies to ameliorate injury, accelerate recovery, or prevent postinjury fibrosis after AKI. Here, we sought to identify candidate compounds that accelerate recovery after AKI by screening for small molecules that increase proliferation of renal progenitor cells in zebrafish embryos. One compound identified from this screen was the histone deacetylase inhibitor methyl-4-(phenylthio)butanoate, which we subsequently administered to zebrafish larvae and mice 24-48 hours after inducing AKI. In zebrafish, treatment with the compound increased larval survival and proliferation of renal tubular epithelial cells. In mice, treatment accelerated recovery, reduced postinjury tubular atrophy and interstitial fibrosis, and increased the regenerative capacity of actively cycling renal tubular cells by decreasing the number of cells in G2/M arrest. These data suggest that accelerating recovery may be a viable approach to treating AKI and provide proof of concept that a screen in zebrafish embryos can identify therapeutic candidates for kidney injury.

Bone marrow plasminogen activator inhibitor-1 influences the development of obesity.

Plasma levels of plasminogen activator inhibitor-1 (PAI-1) are elevated in obesity and correlate with body mass index. The increase in PAI-1 associated with obesity likely contributes to increased cardiovascular risk and may predict the development of type 2 diabetes mellitus. Although adipocytes are capable of synthesizing PAI-1, the bulk of evidence indicates that cells residing in the stromal fraction of visceral fat are the primary source of PAI-1. We hypothesized that bone marrow-derived PAI-1, e.g. derived from macrophages located in visceral fat, contributes to the development of diet-induced obesity. To test this hypothesis, male C57BL/6 wild-type mice and C57BL/6 PAI-1 deficient mice were transplanted with either PAI-1-/-, PAI-1+/-, or PAI-1+/+ bone marrow. The transplanted animals were subsequently fed a high fat diet for 24 weeks. Our findings show that only the complete absence of PAI-1 protects from the development of diet-induced obesity, whereas the absence of bone marrow-derived PAI-1 protects against expansion of the visceral fat mass. Remarkably, there is a link between the PAI-1 levels, the degree of inflammation in adipose tissue, and the development of obesity. Based on these findings we suggest that bone marrow-derived PAI-1 has an effect on the development of obesity through its effect on inflammation.

Bmp2 and Bmp4 exert opposing effects in hypoxic pulmonary hypertension

The bone morphogenetic protein (BMP) type 2 receptor ligand, Bmp2, is upregulated in the peripheral pulmonary vasculature during hypoxia-induced pulmonary hypertension (PH). This contrasts with the expression of Bmp4, which is expressed in respiratory epithelia throughout the lung. Unlike heterozygous null Bmp4 mice (Bmp4(LacZ/+)), which are protected from the development of hypoxic PH, mice that are heterozygous null for Bmp2 (Bmp2(+/-)) develop more severe hypoxic PH than their wild-type littermates. This is associated with reduced endothelial nitric oxide synthase (eNOS) expression and activity in the pulmonary vasculature of hypoxic Bmp2(+/-) but not Bmp4(LacZ/+) mutant mice. Furthermore, exogenous BMP2 upregulates eNOS expression and activity in intrapulmonary artery and pulmonary endothelial cell preparations, indicating that eNOS is a target of Bmp2 signaling in the pulmonary vasculature. Together, these data demonstrate that Bmp2 and Bmp4 exert opposing roles in hypoxic PH and suggest that the protective effects of Bmp2 are mediated by increasing eNOS expression and activity in the hypoxic pulmonary vasculature.

A PTBA small molecule enhances recovery and reduces postinjury fibrosis after aristolochic acid-induced kidney injury

Phenylthiobutanoic acids (PTBAs) are a new class of histone deacetylase (HDAC) inhibitors that accelerate recovery and reduce postinjury fibrosis after ischemia-reperfusion-induced acute kidney injury. However, unlike the more common scenario in which patients present with protracted and less clearly defined onset of renal injury, this model of acute kidney injury gives rise to a clearly defined injury that begins to resolve over a short period of time. In these studies, we show for the first time that treatment with the PTBA analog methyl-4-(phenylthio)butanoate (M4PTB) accelerates recovery and reduces postinjury fibrosis in a progressive model of acute kidney injury and renal fibrosis that occurs after aristolochic acid injection in mice. These effects are apparent when M4PTB treatment is delayed 4 days after the initiating injury and are associated with increased proliferation and decreased G2/M arrest of regenerating renal tubular epithelial cells. In addition, there is reduced peritubular macrophage infiltration and decreased expression of the macrophage chemokines CX3Cl1 and CCL2. Since macrophage infiltration plays a role in promoting kidney injury, and since renal tubular epithelial cells show defective repair and a marked increase in maladaptive G2/M arrest after aristolochic acid injury, these findings suggest M4PTB may be particularly beneficial in reducing injury and enhancing intrinsic cellular repair even when administered days after aristolochic acid ingestion.

Deletion of Fibroblast Growth Factor Receptor 2 from the Peri-Wolffian Duct Stroma Leads to Ureteric Induction Abnormalities and Vesicoureteral Reflux

Purpose Pax3cre-mediated deletion of fibroblast growth factor receptor 2 (Fgfr2) broadly in renal and urinary tract mesenchyme led to ureteric bud (UB) induction defects and vesicoureteral reflux (VUR), although the mechanisms were unclear. Here, we investigated whether Fgfr2 acts specifically in peri-Wolffian duct stroma (ST) to regulate UB induction and development of VUR and the mechanisms of Fgfr2 activity. Methods We conditionally deleted Fgfr2 in ST (Fgfr2ST−/−) using Tbx18cre mice. To look for ureteric bud induction defects in young embryos, we assessed length and apoptosis of common nephric ducts (CNDs). We performed 3D reconstructions and histological analyses of urinary tracts of embryos and postnatal mice and cystograms in postnatal mice to test for VUR. We performed in situ hybridization and real-time PCR in young embryos to determine mechanisms underlying UB induction defects. Results We confirmed that Fgfr2 is expressed in ST and that Fgfr2 was efficiently deleted in this tissue in Fgfr2ST−/− mice at embryonic day (E) 10.5. E11.5 Fgfr2ST−/− mice had randomized UB induction sites with approximately 1/3 arising too high and 1/3 too low from the Wolffian duct; however, apoptosis was unaltered in E12.5 mutant CNDs. While ureters were histologically normal, E15.5 Fgfr2ST−/− mice exhibit improper ureteral insertion sites into the bladder, consistent with the ureteric induction defects. While ureter and bladder histology appeared normal, postnatal day (P) 1 mutants had high rates of VUR versus controls (75% versus 3%, p = 0.001) and occasionally other defects including renal hypoplasia and duplex systems. P1 mutant mice also had improper ureteral bladder insertion sites and shortened intravesicular tunnel lengths that correlated with VUR. E10.5 Fgfr2ST−/− mice had decreases in Bmp4 mRNA in stromal tissues, suggesting a mechanism underlying the ureteric induction and VUR phenotypes. Conclusion Mutations in FGFR2 could possibly cause VUR in humans.

Organ-Specific Defects in Insulin-Like Growth Factor and Insulin Receptor Signaling in Late Gestational Asymmetric Intrauterine Growth Restriction in Cited1 Mutant Mice

Late gestational placental insufficiency resulting in asymmetric intrauterine organ growth restriction (IUGR) is associated with an increased incidence of diabetes, cardiovascular and renal disease in adults. The molecular mechanisms mediating these defects are poorly understood. To explore this, we investigated the mechanisms leading to IUGR in Cited1 knockout mice, a genetic model of late gestational placental insufficiency. We show that loss of placental Cited1 leads to asymmetric IUGR with decreased liver, lung, and kidney sizes and preservation of fetal brain weight. IGF and insulin signaling regulate embryonic organ growth. IGF-I and IGF-II protein and mRNA expression are reduced in livers, lungs, and kidneys of embryonic d 18.5 embryos with IUGR. Decreased IGF-I is associated with reduced activating phosphorylation of the type 1 IGF receptor (pIGF-IR) in the kidney, whereas reduced IGF-II is associated with decreased phosphorylation of the insulin receptor (pIR) in the lung. In contrast, decreased pIR is associated with reduced IGF-I but not IGF-II in the liver. However, pancreatic β-cell mass and serum insulin levels are also decreased in mice with IUGR, suggesting that hepatic IR signaling may be regulated by alterations in fetal insulin production. These findings contrast with observations in IUGR fetal brains in which there is no change in IGF-IR/IR phosphorylation, and IGF-I and IGF-II expression is actually increased. In conclusion, IUGR disrupts normal fetal IGF and insulin production and is associated with organ-specific defects in IGF-IR and IR signaling that may regulate asymmetric IUGR in late gestational placental insufficiency.

Extracellular nucleotide regulation and signaling in cardiac fibrosis

Following myocardial infarction, purinergic nucleotides and nucleosides are released via non-specific and specific mechanisms in response to cellular activation, stress, or injury. These extracellular nucleotides are potent mediators of physiologic and pathologic responses, contributing to the inflammatory and fibrotic milieu within the injured myocardium. Via autocrine or paracrine signaling, cell-specific effects occur through differentially expressed purinergic receptors of the P2X, P2Y, and P1 families. Nucleotide activation of the ionotropic (ligand-gated) purine receptors (P2X) and several of the metabotropic (G-protein-coupled) purine receptors (P2Y) or adenosine activation of the P1 receptors can have profound effects on inflammatory cell function, fibroblast function, and cardiomyocyte function. Extracellular nucleotidases that hydrolyze released nucleotides regulate the magnitude and duration of purinergic signaling. While there are numerous studies on the role of the purinergic signaling pathway in cardiovascular disease, the extent to which the purinergic signaling pathway modulates cardiac fibrosis is incompletely understood. Here we provide an overview of the current understanding of how the purinergic signaling pathway modulates cardiac fibroblast function and myocardial fibrosis.

Delayed treatment with PTBA analogs reduces postinjury renal fibrosis after kidney injury

No therapies have been shown to accelerate recovery or prevent fibrosis after acute kidney injury (AKI). In part, this is because most therapeutic candidates have to be given at the time of injury and the diagnosis of AKI is usually made too late for drugs to be efficacious. Strategies to enhance post-AKI repair represent an attractive approach to address this. Using a phenotypic screen in zebrafish, we identified 4-(phenylthio)butanoic acid (PTBA), which promotes proliferation of embryonic kidney progenitor cells (EKPCs), and the PTBA methyl ester UPHD25, which also increases postinjury repair in ischemia-reperfusion and aristolochic acid-induced AKI in mice. In these studies, a new panel of PTBA analogs was evaluated. Initial screening was performed in zebrafish EKPC assays followed by survival assays in a gentamicin-induced AKI larvae zebrafish model. Using this approach, we identified UPHD186, which in contrast to UPHD25, accelerates recovery and reduces fibrosis when administered several days after ischemia-reperfusion AKI and reduces fibrosis after unilateral ureteric obstruction in mice. UPHD25 and 186 are efficiently metabolized to the active analog PTBA in liver and kidney microsome assays, indicating both compounds may act as PTBA prodrugs in vivo. UPHD186 persists longer in the circulation than UPHD25, suggesting that sustained levels of UPHD186 may increase efficacy by acting as a reservoir for renal metabolism to PTBA. These findings validate use of zebrafish EKPC and AKI assays as a drug discovery strategy for molecules that reduce fibrosis in multiple AKI models and can be administered days after initiation of injury.

Impact of cardiac-specific expression of CD39 on myocardial infarct size in mice

Aims: Prior work suggests that ischemic preconditioning increases the level of CD39 in the heart and contributes to cardiac protection. Therefore, we examined if targeted cardiac expression of CD39 protects against myocardial injury. Main methods: Mice with cardiac‐specific expression of human CD39 (&agr;MHC/hCD39‐Tg) were generated, characterized and subjected to left coronary artery ischemia‐reperfusion injury and infarct size at 24 h following injury quantified. Key findings: &agr;MHC/hCD39‐Tg mice have increased in cardiac ATPase and ADPase activity compared to WT littermates. The increased activity in &agr;MHC/hCD39‐mice was inhibited by the CD39 antagonist sodium polyoxotungstate (POM‐1). Measurement of basal cardiac function by echocardiography revealed that &agr;MHC/hCD39‐Tg mice have a lower resting heart rate and increased stroke volume. In response to myocardial ischemia, systolic and diastolic function was better preserved in &agr;MHC/hCD39‐Tg compared to WT mice. Comparison of Tau also revealed preserved cardiac relaxation during ischemia in &agr;MHC/hCD39‐Tg hearts. Assessment of myocardial infarct size in response to 60 min of ischemia and 24 h of reperfusion demonstrated a significant reduction in infarct size in &agr;MHC/hCD39‐Tg hearts. Analysis of isolated cardiomyocytes revealed no basal difference in calcium transients between WT and &agr;MHC/hCD39‐Tg cardiomyocytes. However, in response to isoproterenol stimulation, there was a trend toward lower calcium transients in &agr;MHC/hCD39 cardiomyocytes suggesting less calcium accumulation in response to metabolic stress. Significance: Cardiac‐specific expression of CD39 reduces myocardial dysfunction and infarct size following ischemia‐reperfusion injury. Increasing nucleotidase expression in the heart may be a novel approach to protect the heart from ischemic injury.

Role of the CD39/CD73 Purinergic Pathway in Modulating Arterial Thrombosis in Mice

Objective—Circulating blood cells and endothelial cells express ectonucleoside triphosphate diphosphohydrolase-1 (CD39) and ecto-5′-nucleotidase (CD73). CD39 hydrolyzes extracellular ATP or ADP to AMP. CD73 hydrolyzes AMP to adenosine. The goal of this study was to examine the interplay between CD39 and CD73 cascade in arterial thrombosis. Approach and Results—To determine how CD73 activity influences in vivo thrombosis, the time to ferric chloride–induced arterial thrombosis was measured in CD73-null mice. In response to 5% FeCl3, but not to 10% FeCl3, there was a significant decrease in the time to thrombosis in CD73-null mice compared with wild-type mice. In mice overexpressing CD39, ablation of CD73 did not inhibit the prolongation in the time to thrombosis conveyed by CD39 overexpression. However, the CD73 inhibitor &agr;-&bgr;-methylene-ADP nullified the prolongation in the time to thrombosis in human CD39 transgenic (hC39-Tg)/CD73-null mice. To determine whether hematopoietic-derived cells or endothelial cell CD39 activity regulates in vivo arterial thrombus, bone marrow transplant studies were conducted. FeCl3-induced arterial thrombosis in chimeric mice revealed a significant prolongation in the time to thrombosis in hCD39-Tg reconstituted wild-type mice, but not on wild-type reconstituted hCD39-Tg mice. Monocyte depletion with clodronate-loaded liposomes normalized the time to thrombosis in hCD39-Tg mice compared with hCD39-Tg mice treated with control liposomes, demonstrating that increased CD39 expression on monocytes protects against thrombosis. Conclusions—These data demonstrate that ablation of CD73 minimally effects in vivo thrombosis, but increased CD39 expression on hematopoietic-derived cells, especially monocytes, attenuates in vivo arterial thrombosis.