Doctor of Philosophy, University of Toledo, 2023, Chemistry

Elevated levels of endogenous cardiotonic steroids (CTS), such as telocinobufagin (TCB) and marinobufagin (MBG), are observed in patients with chronic kidney disease (CKD). The characteristic six-membered δ-lactone ring structure of CTS is critical for binding to the Na+ /K+-ATPase and subsequent signaling. Paraoxonases (PONs) belong to a family of hydrolytic enzymes that can regulate levels of δ-lactone ring structures, like those found in CTS, rendering them inactive. Clinical studies have reported decreased lactonase activity of PONs in CKD patients and its association with cardiovascular morbidity and mortality. PON's activity is diminished in CKD, yet it is unclear whether it is mechanistically linked to increased CTS levels. This research aimed to enhance the knowledge regarding CTS by exploring their regulation in the body and association with PON enzymes, whose exact physiological substrate(s) is/are still unknown. To bridge these knowledge gaps, this research describes novel methods for extraction, detection, and quantification of CTS using highly specific, sensitive, and accurate analytical techniques, such as ultrahigh-performance liquid chromatography-mass spectrometry (UHPLC-MS) as well as HPLC-photodiode array (PDA). In the first project, a protocol was developed for the extraction, detection and quantification of TCB and MBG. Solid-phase extraction (SPE) with hydrophilic-lipophilic balanced (HLB) cartridges was used for the extraction of CTS from an aqueous solution. The methods were optimized to extract and quantify TCB and MBG from the urine of rats with different genotypes; wild type (WT) and PON3 knockout (KO) subjected to different diets (normal chow and high salt) for 8 weeks. The high salt-treated rats serve as a model for studying CKD, while the wild type rats on normal chow act as controls. The results showed that the urine of SS-PON3 KO rats contained higher CTS levels than urine from SS-WT rats for both control and high-salt samples, sug (open full item for complete abstract)

Committee: Dragan Isailovic (Committee Chair); David Kennedy (Committee Member); Emanuela Gionfriddo (Committee Member); Jianglong Zhu (Committee Member) Subjects: Biomedical Research; Chemistry

Master of Science (MS), Wright State University, 2018, Pharmacology and Toxicology

The primary function of Na+/K+ATPase (NKA) discovered by Jens Christian Skou in 1958, through electrogenic exchange of 3 Na+ for 2 K+ ions (Robert L Post, 1958), is the homeostatic preservation of their electrochemical gradient across the plasma membrane (PM) of almost all species and their derived cell lines. Cardiotonic steroids (CTS), such as cardenolides or bufadienolides, are well established inhibitors of NKA. Fox glove plant extracts containing CTS, including digitalis, were first described by William Withering (1744-91) for their positive inotropic effect in heart failure patients. Later research identified the mechanism of their action through direct inhibition of NKA with simultaneous cytosolic Na+ accumulation and due to reversal of the Na+/Ca2+ exchanger located in series with NKA in the plasma membrane. Subsequent increases in cytoplasmic Ca2+ levels lead to higher contractility of the heart, the inotropic effect of CTS. The Strophantus gratus-derived CTS ouabain is experimentally widely used. During the past two decades, ouabain binding to NKA, resulting in the here so coined NKA-ouabain receptor complex (NORC), has been shown to elicit at least two signaling pathways involving protein kinases with sequential phosphorylation/ transphosphorylation reactions leading to transcriptional upregulation of proteins iv involved in cell proliferation, survival, and death. More recent studies suggest NORC internalization through the plasmalemmal membrane and its cytosolic appearance. Mechanisms of NORC internalization and ensuing putative actions of NKA or ouabain or both have yet to be identified. In the present work, it was hypothesized that during its membrane to cytosol descent, NORC utilizes a variety of cytosolic and cytoskeletal proteins through protein/protein interactions as an escalator to reach endosomes and lysosomes for subsequent breakup into its NKA receptor and ligand ouabain. NORC internalization would then serve as an alt (open full item for complete abstract)

Committee: Peter Lauf M.D. (Advisor); Norma Adragna Ph.D. (Committee Member); Ulas Sunar Ph.D. (Committee Member) Subjects: Pharmacology; Toxicology

PhD, University of Cincinnati, 2018, Arts and Sciences: Chemistry

A new analytical method was designed to quantify the activity of the Na+, K+ ATPase in skeletal muscle and red blood cells based on ICP-MS-MS. This new approach will aid in determining the physiological effects of novel endogenous cardiotonic steroids extracted from pig skeletal muscle. The Na+, K+ ATPase is a vital enzyme in all eukaryotic cells. The Na+, K+ ATPase is also the receptor for cardiotonic steroids, a class of compounds that are used in the treatment of heart failure as well as many other medical conditions. Endogenous cardiotonic steroids are present at low levels in tissues and blood, and these compounds play a significant role in blood pressure regulation and cardiac function. The presence of uncharacterized cardiotonic steroids in tissues presents a relevant area for research. The first part of my dissertation focuses on metal ion transport by the Na+, K+ ATPase in cells which were quantified by ICP-MS-MS. The activity of the Na+, K+ ATPase in mouse skeletal muscles and human red blood cells was measured based on the amount of Rb, as a K analog and K present upon quantification by ICP-MS-MS. The previous method used to quantify Rb and measure Na+, K+ ATPase activity in tissues and cells required the use of radioactive materials. The instrument used for the previous method is called a scintillation counter which can detect and measure the ionizing radiation from 86Rb. The new approach using ICP-MS-MS does not require the use of radioactive 86Rb. Instead, natural abundance 85Rb and 87Rb can be used to quantify Rb within the cells to determine the activity of the Na+, K+ ATPase. This new method helps to study physiological processes that are metal-dependent and can be used for various cell types and conditions. The second part of my dissertation focuses on ICP-MS-MS analysis of micro-sized biological samples with heteroatoms as an internal tag for mass-free quantification of selected elements. ICP-MS-MS has been used for elemental analysis at t (open full item for complete abstract)

Committee: Julio Landero Figueroa Ph.D. (Committee Chair); Edward Merino Ph.D. (Committee Chair); Judith Heiny Ph.D. (Committee Member); Pearl Tsang Ph.D. (Committee Member) Subjects: Analytical Chemistry

Master of Science in Biomedical Sciences (MSBS), University of Toledo, 2017, Biomedical Sciences (Bioinformatics and Proteomics/Genomics)

Introduction: Cardiotonic steroids (CTSs) are steroid hormones which are elevated in chronic kidney disease (CKD). The 2-pyrone ring structure of CTS is critical for its binding to the Na+/K+-ATPase and subsequent initiation of profibrotic signaling which promotes tissue fibrosis and organ dysfunction underlying cardiac and renal disease. Paraoxonases (PONs) are a family of hydrolytic enzymes which can regulate levels of 2-pyrone structures like those found in CTS, and we have recently discovered that the activity of these enzymes is diminished in setting such as CKD where CTSs are elevated. Hypothesis: We hypothesized that dysregulated expression in the PON gene family is associated with renal disease and that PONs, via their hydrolytic activity, participate in the metabolism and regulation of CTSs. Methods/ Results: We used molecular, biochemical, and bioinformatic approaches to study how PONs regulate CTSs in the setting of renal disease. We first performed a meta-analysis (accessed from NCBI's Gene Expression Omnibus (GEO) Datasets) of normalized PON isoform gene expression from whole genome microarray data of both peripheral blood mononuclear cells (PBMC's, n=4 studies, 174 total patients) and renal biopsy samples (n=4 studies, 207 total patients), in case-control studies of patients with chronic kidney disease (CKD stage 2-5) and the respective non-CKD controls. From these studies, we noted that PON-1 gene expression was significantly decreased in PBMCs of CKD (Stage 2-3) patients vs non-CKD controls (0.8±0.03 vs 1.0±0.03, p<0.05), however in renal biopsy specimens PON-1 (2.7±0.42 vs 1.0±0.02, p<0.05) and PON-2 (1.5±0.06 vs 1.0±0.04, p<0.01) was significantly increased in CKD (Stage 2-3) patients vs non-CKD controls. We also searched for the genes which are highly correlated such as GADD45B, STK26, IFITM2///IFITM1, ZNF418, etc. with PON genes in the setting of chronic kidney disease. Next, we measured circulating PON-1 protein (ELISA) and activity (as mea (open full item for complete abstract)

Committee: David Kennedy (Committee Chair); Sadik Khuder (Committee Member); Bruce Levison (Committee Member) Subjects: Bioinformatics; Biomedical Research

Doctor of Medicine and Doctor of Philosophy, University of Toledo, 2014, College of Medicine

Cardiotonic steroids (CTS), including ouabain and marinobufagenin, have a natriuretic effect through their action on the Na+/K+-ATPase in the kidney. In addition to a role in inducing salt-sensitive hypertension, they are also implicated in insulin regulation. CarcinoEmbryonic Antigen Cell Adhesion Molecule (CEACAM) protein, a known mediator of insulin clearance via its interaction with the insulin receptor (IR), among its other functions, is also expressed in the kidney, and involved in the development of insulin resistance. We investigate a possible interaction between cardiotonic steroids and insulin in signaling through the sodium pump toward the development of insulin resistance and salt-sensitive hypertension. The sodium pump, insulin receptor, epidermal growth factor receptor (EGFR), and CEACAM1 are expressed on the basolateral aspect of polarize renal proximal tubule LLC-PK1 cells, where they can interact to facilitate intracellular signaling. Cardiotonic steroids and insulin, both of which can signal through the Na+/K+-ATPase, stimulated decreased plasmalemmal expression, with corresponding increase in early endosomal accumulation, of CEACAM1 protein. EGFR and IR showed similar changes with exogenous ouabain or insulin stimulation, though these changes were not synergistic. Thus, in addition to its role in insulin clearance in the liver, CEACAM1 could also play a role in cardiotonic steroid-induced natriuresis and salt-sensitive hypertension in the kidney. This was supported by increased salt-sensitive hypertension in those mice lacking CEACAM2, a homologous and possibly functionally redundant protein present along with CEACAM1 in mice. If as predicted, the incidence of diabetes does indeed increase in the future, associated diseases such as chronic renal failure may also have a greater impact. Taken together, these data provide molecular insight into a well-documented clinical association between chronic renal failure and insulin resistance. CEACAM, in ad (open full item for complete abstract)

Committee: Deepak K. Malhotra (Committee Chair); Joseph I. Shapiro (Committee Member); Sonia M. Najjar (Committee Member); David R. Giovannucci (Committee Member); Jiang Tian (Committee Member) Subjects: Biomedical Research; Physiology

PhD, University of Cincinnati, 2010, Medicine: Systems Biology and Physiology

Chronic heart failure (CHF) remains a major public health problem, despite advances in the prevention and treatment of cardiovascular diseases. Cardiotonic steroids (CS), such as digoxin, are used therapeutically to treat CHF, and are mechanistically based on altered activity of its target receptor, the alpha subunit of the Na,K-ATPase pump. In addition to its conventional role as an ion transporter, recent evidence suggests that the sodium pump may also act as a signal transducer upon CS binding and induce non-proliferative cardiac growth, implicating a role in the development of cardiac hypertrophy and progressive failure of the heart. Moreover, elevated concentrations of endogenous CS have been detected in diverse clinical situations such as chronic renal failure, congestive heart failure and essential hypertension, however, the exact (patho) physiological role of endogenous CS is not fully understood. The hypothesis of this dissertation was that hypertrophic response to pressure overload would be aggravated in mutant mice with an ouabain-sensitive α1 Na,K- ATPase (NKA) isoform in response to endogenous CS. To test this hypothesis, transverse aortic constriction (TAC) surgery was performed in α1-resistant/α2-resistant (α1R/R α2 R/R), α1-sensitive/α2-resistant (α1S/S α2 R/R), and α1-resistant/α2-sensitive mice (α1R/R α2 S/S, wild type) to experimentally produce chronic pressure overload induced left ventricular (LV) hypertrophy. We have evidence that mutant mice with altered “ouabain-sensitivity” of the various α-subunits of the Na,K-ATPase represent useful models for investigating the (patho)physiological effects of endogenous caredenolides and bufadienolides and the underlying molecular mechanisms that involve specific Na,K-ATPase α-subunits. Echocardiography was performed after TAC to assess post-TAC cardiac structure and function. At 4 weeks α1 S/S α2 R/R mice showed a pronounced increase in LV weight and wall thickness with extensive perivascular and replacem (open full item for complete abstract)

Committee: John Lorenz PhD (Committee Chair); Gary Edward Shull PhD (Committee Member); Nelson Horseman PhD (Committee Member); Jo El Schultz PhD (Committee Member); Judith Heiny PhD (Committee Member) Subjects: Physiological Psychology

Doctor of Philosophy (PhD), University of Toledo, 2012, College of Medicine

Cardiotonic steroids such as marinobufagenin (MBG) and ouabain are specific ligands for the Na/K-ATPase and represent a relatively new class of steroid hormones. Uremic cardiomyopathy is characterized by a decrease in diastolic function, left ventricular hypertrophy, oxidant stress, and both cardiac and renal fibrosis. We have shown that MBG, signaling through the Na/K-ATPase, causes many of the adverse pathological effects of experimental uremic cardiomyopathy induced by 5/6th nephrectomy (PNx) in the rat. The goal of this dissertation is to describe some of the manipulations we have performed in order to provide potential therapies for the treatment of uremic cariomyopathy. Specifically, we show that treatment with an anti-MBG antibody drastically reduces cardiac fibrosis in PNx animals. Treatment with rapamycin (an mTOR inhibitor) produced similar effects with the added benefit of reducing circulating MBG in these animals. In addition, we show that ischemic renal disease is accompanied with elevated levels of the platelet activation marker soluble CD40 ligand (sCD40L), and its soluble receptor, CD40, may predict outcomes in this disease state. Data in our PNx model suggests a role for proximal tubular CD40 activation contributing to the development of renal fibrosis, which may be potentiated by cardiotonic steroid signaling through the Na/K-ATPase.

Committee: Joseph Shapiro MD (Committee Chair); Christopher Cooper MD (Committee Member); Deepak Malhotra MD, PhD (Committee Member); Zijian Xie PhD (Committee Member); Jiang Liu PhD (Committee Member) Subjects: Biomedical Research

Doctor of Philosophy in Biomedical Sciences (Ph.D.), University of Toledo, 2010, College of Medicine

Our laboratory previously reported that ouabain decreases the transcellular sodium transport in LLCPK1 cell line by mechanisms other than affecting intracellular sodium concentration (Liu J 2002). We utilized for our experiments LLCPK1 cell line, which is an epithelial cells derived from pig renal proximal tubules. When cell were treated for 12 hours with low dose of ouabain (50nM and 100 nM), the intracellular sodium concentration was not affected. Moreover, NHE3, the major apical sodium transporter in proximal tubules, was significantly downregulated on mRNA level, as measured by StaRT-PCR. NHE3 protein was also downregulated when measured by westernblot. Total NHE activity was also decreased when measured by intracellular PH recovery after acidification. Ouabain-induced downregulation of NHE3 was blocked after inhibiting c-src or PI3K with PP2 or wortmannin respectively. Both c-src and PI3K are essential part of Na/K ATpase signaling cascade. We utilized transiently transfected cells with NHE3 promoter constructs in luceferase reporter vector to map the response elements. We mapped the ouabain response elements on NHE3 promoter to be (-450 to -1194). Furthermore, Ouabain-induced downregulation of NHE3 promoter activity was blocked in C2-9 cell line (siRNA-mediated caveolin-1 depleted cell line). We used C2-9 cell line to confirm the role of pump endocytosis in the ouabian signaling cascade to regulate NHE3 expression. Our laboratory identified that ouabain decreases the binding of transcription factors TR and Sp1 to their cognate cis-elements on the promoter. Our group utilized protein/DNA array analysis, chromatin immunoprecipitation and electrophoretic mobility shift assay (EMSA) to confirm the decrease of the transcription factors binding induced by ouabain. We concluded that ouabain chronically downregulates NHE3 expression and acitivty in proximal tubule cells by endocytosis-mediated signaling cascade from Na/K ATPase. The signaling pathway involves c-src (open full item for complete abstract)

Committee: Joseph Shapiro (Committee Chair); Sonia Najjar (Committee Member); Deepak Malhotra (Committee Member); Jiang Liu (Committee Member); Zijian Xie (Committee Member) Subjects: Biomedical Research

Master of Science in Biomedical Sciences (MSBS), University of Toledo, 2008, College of Graduate Studies

We previously reported that cardiotonic steroids stimulate collagen synthesis by cardiac fibroblasts in a process that involves signaling through the Na/K-ATPase pathway. In this study, we examined the effect of cardiotonic steroids on dermal fibroblasts collagen synthesis and on wound healing. Increased collagen expression by human dermal fibroblasts was noted in response to the cardiotonic steroid, marinobufagenin, in a dose and time dependent-fashion. Next, we examined the effect of digoxin, ouabain and marinobufagenin on the rate of wound closure in an in vitro model where human dermal fibroblasts cultures were wounded with a pipette tip and monitored by digital microscopy. Finally, we administered digoxin in an in vivo wound healing model, using olive oil as a vector. This application significantly accelerated in vivo wound healing in rats wounded with an 8 mm biopsy cut. Increased collagen accumulation was noted 9 days after wounding (both p < 0.01). These data suggest that cardiotonic steroids induce increases in collagen synthesis by dermal fibroblasts, and accelerate wound healing.

Committee: Joseph Shapiro MD (Committee Chair); Sonia Najjar PhD (Committee Member); Zi-Jian Xie PhD (Committee Member); Deepak Malhotra MD, PhD (Committee Member) Subjects: Molecular Biology; Pharmacology; Surgery

Doctor of Philosophy in Medical Sciences (Ph.D.), University of Toledo, 2008, College of Graduate Studies

It has been recognized that patients with chronic renal failure eventually develop diastolic dysfunction, cardiac hypertrophy and systemic oxidant stress along with increases in circulating concentrations of the cardiotonic steroid, marinobufagenin (MBG) in uremic cardiomyopathy. Because of this, we performed 5/6th partial nephrectomy in rats to study experimental renal failure (PNx), MBG infusion, PNx after immunization against MBG, and concomitant PNx and adrenalectomy. We also studied 5⁄6th partial nephrectomy as a potential experimental renal failure model in mice. Next, we speculated a relationship between decreases Fli-1 expression and increases in collagen production following exposure to MBG. Therefore, we examined Fli-1 knockdown mice and compared it to wild type mice. Physiological measurements with a Millar catheter and immunohistochemistry were performed. In vitro studies were then pursued with cultured isolated cardiac fibroblasts, human dermal fibroblasts, as well as a cell line derived from renal fibroblasts. First, in rats, we observed that PNx after immunization against MBG as well as concomitant PNx and adrenalectomy had similar blood pressure as PNx but less cardiac hypertrophy, diastolic dysfunction, and cardiac fibrosis. Second, in mice, the 5⁄6 nephrectomy resulted in impairment of both active and passive left ventricular relaxation at four weeks as well as progressive fibrosis in the heart. Third, the Fli-1 knockdown mice showed greater amounts of cardiac collagen expression and fibrosis compared to wild type before and after 4 weeks of experimental renal failure induced by 5/6th nephrectomy. We realized that stimulation of cultured cardiac fibroblasts with MBG could be prevented by administration of inhibitors of tyrosine phosphorylation, Src activation, EGFR transactivation, and N-acetyl cysteine. Furthermore, in response to MBG, decreases in nuclear Fli-1 accompanied increases in procollagen expression. Finally, we observed that exposure o (open full item for complete abstract)

Committee: Joseph Shapiro MD (Committee Chair); Jiang Liu PhD (Committee Member); Sonia Najjar PhD (Committee Member); Zi-Jian Xie PhD (Committee Member); Sandrine Pierre PhD (Committee Member) Subjects: Pharmacology; Surgery

Master of Science in Biomedical Sciences (MSBS), University of Toledo, 2008, College of Graduate Studies

Cardiotonic steroids such as MBG have been shown to induce cardiac fibrosis. MBG contributes to renal fibrosis as well by inducing epithelial to mesenchymal transition (EMT). The porcine kidney cell line, LLC-PK1, acquires mesenchymal features such as a fibroblast-like phenotype, scattering and invasive properties and increased expression of mesenchymal proteins fibronectin and vimentin in a time and concentration dependent fashion. To examine the mechanisms which are operant, Western blotting and immunostaining were used. LLC-PK1 cells were grown to complete confluence and treated with 100 nM of MBG for 72-96 hours. At this concentration, MBG induces profound EMT (more than 50% of cells show fibroblast-like morphology; scattered cells occupy more than 80% of the cell- grown surface). It decreases the expression of epithelial proteins-E-cadherin, occludin and claudin-1 and increases the expression of mesenchymal proteins-vimentin and fibronectin by two fold. These alterations of LLC-PK1 cells are accompanied by 1) translocation of transcription factor Snail into nuclei after 72-96 hours post treatment and 2) de novo expression of transcription factor LEF1 at 72 hours post treatment, and its accumulation in the nuclei at 96 hours. Interestingly, distribution of the LEF1 co-activator, beta-catenin, does not change with MBG treatment. ROS scavenger NAC completely prevents morphological alteration of LLC-PK1 cells and inhibits the up-regulation of fibronectin caused by MBG. The Src inhibitor, PP2, shows only partial attenuation of the morphological alterations of LLC-PK1 cells induced by MBG but it completely abolishes MBG-induced up regulation of fibronectin. This study indicates that MBG induces EMT in LLC-PK1 cells and the two transcription regulators of the EMT transcriptome, Snail and LEF1 in a process requiring reactive oxygen species.

Committee: Joseph Shapiro MD (Committee Chair); Deepak Malhotra PhD (Committee Member); Zi-Jian Xie PhD (Committee Member) Subjects: Molecular Biology

Doctor of Philosophy in Medical Sciences (Ph.D.), University of Toledo, 2006, College of Graduate Studies

Patients with chronic renal failure develop a “uremic” cardiomyopathy characterized by diastolic dysfunction, cardiac hypertrophy and systemic oxidant stress. Patients with chronic renal failure also are known to have increases in the circulating concentrations of marinobufagenin (MBG), a cardiotonic steroid. On this background, we investigated a clinical population of patients with ischemic renal disease in order to examine the implications of stabilizing or improving renal function as it related to the effects on cardiac morbidity and mortality. We then employed the 5/6th nephrectomy model (PNX) in the rat in order to examine the molecular mechanisms by which chronic renal failure contributes to cardiac abnormalities and to examine the role of MBG in the systemic oxidant stress state and cardiac changes seen with experimental uremia. Finally, we examined the reactive oxygen/nitrogen (ROS/RNS) dependent mechanisms by which cardiotonic steroids modulate cardiac function. First, we observed that in patients with renal artery stenosis undergoing stent therapy,baseline renal insufficiency is associated with an increased incidence of morbidity and mortality, independent of other baseline clinical factors. Importantly, improvement in renal function appears to be associated with increased survival. Next, using the PNX model we observed that chronic renal failure leads to alterations in cardiac gene expression and produced alterations in cardiac calcium cycling and contractile function in the rat. Administration of MBG caused comparable increases in plasma MBG, blood pressure, cardiac weight and diastolic dysfunction as PNX at 4 wk. Decreases in the 265 expression of the cardiac sarcoplasmic reticulum ATPase (SERCA2a), cardiac fibrosis and systemic oxidant stress were observed with both MBG infusion and PNX, while active immunization against MBG attenuated these changes without affecting blood pressure. Finally, acute administration of the cardiotonic steroid ouabain modul (open full item for complete abstract)

Committee: Joseph Shapiro, M.D. (Advisor) Subjects: