• NAE1-MEDIATED NEDDYLATION IS REQUIRED FOR POSTNATAL LIVER DEVELOPMENT AND FUNCTION

      Sahay, Khushboo; Department of Physiology (7/26/2018)
      Liver disease is an important health concern and a significant source of morbidity and mortality in the United States and worldwide. NEDD8 (neural-precursor-cell-expressed developmentally down-regulated 8) is a novel ubiquitin-like protein modifier. The conjugation of NEDD8 to target proteins, termed neddylation, requires NEDD8 specific E1, E2 and E3 ligases. Neddylation participates in various cellular processes. However, whether neddylation regulates liver development and function is completely unknown. We created mice with hepatocyte specific deletion of NAE1, a subunit of the only NEDD8 E1 enzyme, and identified that they display severe hepatomegaly, hypertriglyceridemia, and hypercholesterolemia from 10 days after birth. By postnatal 14 days, their liver cytoarchitecture is completely disrupted, along with formation of numerous biliary cysts, fibrosis and hypoglycemia, which ultimately result in liver failure and premature death by 6 weeks. Mechanistically, NAE1 deficiency in hepatocytes caused reduced hepatocytespecific gene expression but increased biliary/oval cell gene expression in liver. In vitro, NAE1 inhibition by MLN4924 and CRISPR/Cas9-mediated NAE1 deletion in HepG2 cells recapitulated in vivo findings with repressed expression of hepatocyte specific genes but elevated biliary/oval cell gene expression. Together, these data highlight an essential role for neddylation in regulating hepatocyte lineage commitment and function as well as polycyst formation through trans/de-differentiation of hepatocytes.
    • EFFECTS OF SODIUM BICARBONATE ON GLUCOSE HOMEOSTASIS AND BLOOD PRESSURE IN CHRONIC KIDNEY DISEASE

      Mannon, Elinor; Department of Philosophy (Augusta University, 2021-10)
      Sodium bicarbonate (NaHCO3) is a therapeutic used in chronic kidney disease (CKD). NaHCO3 is typically used to treat metabolic acidosis, but clinical studies have indicated that NaHCO3 supplementation may slow CKD progression. As such, NaHCO3 is now given to patients with CKD to slow the decline of glomerular filtration rate. However, the consequences of chronic NaHCO3 supplementation in CKD remain unclear. Acidosis has been associated with insulin resistance, and correction of acidosis with NaHCO3 was reported to improve insulin sensitivity. Our goal in Aim 1 was to determine whether acid and alkali loading would promote loss of acid-base homeostasis and consequently decrease insulin sensitivity. We determined that the blood glucose response to insulin is enhanced following renal mass reduction, and that this response is not reversed by an acidosis. Additionally, the development of an alkalosis did not impair the blood glucose response to insulin. Alkali can promote potassium (K+) wasting, and an association between K+ wasting and insulin resistance has been identified in clinical and basic science research. Our goal in Aim 2 was to identify whether chronic NaHCO3 treatment may promote loss of insulin sensitivity through effects on K+ status. We determined that chronic NaHCO3 treatment impairs insulin sensitivity when combined with other K+ wasting stimuli. K+ deprivation alone also impaired the blood glucose response to insulin, however these impairments in insulin sensitivity were not directly related to decreases in intracellular [K+]. Salt-sensitivity increases as functional renal mass declines, and chronic sodium (Na+) loading with NaHCO3 may contribute to hypertension in patients with CKD. Our goal in Aim 3 was to investigate whether NaHCO3 loading promotes similar levels of Na+ and volume retention, and hypertension as sodium chloride (NaCl) loading does in a rat model of CKD. We found that NaHCO3 was pro-hypertensive, but to a lesser degree than NaCl, despite similar amounts of Na+ and volume retention. From these studies we concluded that NaHCO3 does not improve insulin sensitivity through its effects on acid-base status. Further, access to dietary K+ may improve insulin sensitivity with chronic NaHCO3 treatment. Finally, NaHCO3 can promote hypertension in CKD.
    • Predictive Inference for Linear and Circular Concomitants with Biomedical Applications

      Howie, Melissa; Department of Philosophy (Augusta University, 2021-07)
      Let (X_i, Y_i), for i=1,...,n, be a random sample from a bivariate distribution. If the sample is ordered with respect to one of the variables, say X, then the rth ordered X-value is called the rth order statistic and is denoted X_{r:n}. The Y-value corresponding to this value is called the concomitant of the rth order statistic and is denoted Y_{[r:n]}. In biomedical research, there is an interest in predicting the concomitant variable corresponding to the rth order statistic of the other variable. For example, one may be interested in predicting the time at which a patient has the peak blood pressure or the mercury level in fish where the water is most polluted. One such distribution of interest is the bivariate exponential conditionals distribution (BEC), whose conditional distributions are both exponential. The asymptotic predictive distribution of the concomitants of order statistics from the BEC is derived. The results are used in a prediction problem involving the mercury concentration in largemouth bass sampled from Florida lakes, as a function of surface water pollution level. Clinicians are often confronted with data such that one variable is linear and the other variable is circular, i.e., measured as an angle. A particular linear-circular distribution of interest is the exponential circular normal distribution. The predictive distribution of concomitants of order statistics from the exponential circular normal distribution is derived. The results are applied to predicting the future value of time at maximum heart rate in subjects from the Augusta Heart Study, a longitudinal study of normotensive children with verified family histories of cardiovascular diseases (e.g., hypertension and premature myocardial infarction).
    • MODELING THE SIMULTANEOUS EFFECTS OF COPY NUMBER VARIATION AND METHYLATION ON GENE EXPRESSION USING NEXT GENERATION SEQUENCING DATA

      Claussen, Henry; Department of Physiology (Augusta University, 2021-07)
      The collection and order of nucleobases in a strand of DNA, called the primary sequence, is one of the most important pieces of information in the study of the human body. The proteins which regulate all biological functions in the body are synthesized based on the structure of the DNA molecule. The next generation sequencing (NGS) process of sequencing RNA transcripts, known as RNA-seq, has become a powerful alternative to traditional microarray technology. NGS is used to measure the levels of gene expression, detect structural DNA variations from the human reference genome, and uncover the epigenetic modifications of methylation. Despite its prevalence in genetic research, RNA-seq data suffers from the statistical complication known as ”large p small n” where the predictor variables greatly outnumber the subjects in a study. In this research we propose combining all three types of data into a multivariate linear model. With the implementation of a variable selection process for preliminary dimension reduction and the application of a Group LASSOapproach, we hope to reduce the complexity and dimension of NGS data to a manageable and, most importantly, interpretable level. Changes in gene expression levels have been linked with the development of harmful diseases such as cancer. A successful model will provide insight on the simultaneous effects that methylation and structural variation have on gene expression in the body.
    • A NOVEL NETWORK BASED LINEAR MODEL FOR ENRICHMENT OF SYNERGISTIC DRUG COMBINATIONS

      Li, Jiaqi; Department of Physiology (Augusta University, 2021-07)
      Drug combination therapies can improve drug efficacy, reduce drug dosage, and overcome drug resistance with respect to cancer treatments. Current research strategies to determine which drug combinations have a synergistic effect rely mainly on clinical or empirical experience and screening predefined pools of drugs. Given the number of possible drug combinations, the speed and scope to find new drug combinations are very limited using these methods. Due to the exponential growth in these combinatorials, it is difficult to test all possible outcomes in the lab. Several large-scale public genomic and phenotypic resources that provide data from single drug-treated cells as well as data from small molecules deliver a wealth of cellular response information. This data gives opportunity to overcome limitations of the current methods. The development of a new strategy for advanced data processing and analysis that includes a computational prediction algorithm is highly desirable. Because of this, a program was written that predicts synergistic drug combinations using gene regulatory network knowledge and an operational module unit (OMU) system generated from single drug genomic and phenotypic data. As a proof of principle, we applied the pipeline to a group of anticancer drugs and demonstrated how the algorithm could help researchers efficiently find possible synergistic drug combinations using single drug data to evaluate all possible drug pairs.
    • DEFINING THE ROLE OF AQUEOUS HUMOR PROTEOME IN GLAUCOMATOUS OPTIC NEUROPATHY

      Kodeboyina, Sai Karthik; Department of Physiology (Augusta University, 2021-05)
      Aqueous humor (AH) is a fluid in the anterior and posterior chambers of the eye that contains proteins associated with vision disorders including glaucoma. We performed comprehensive characterization of AH proteins and evaluated their association with optic nerve and visual field changes in glaucoma patients. AH reference database was developed to include proteomic and clinical information from cataract and glaucoma patients. Aqueous humor samples from 251 cataract and glaucoma patients were analyzed using Liquid-Chromatography Mass spectrometry (LC-MS/MS). Retinal nerve fiber layer (RNFL) thickness was evaluated with the SPECTRALIS Tracking Laser Tomography. Optic nerve head imaging was performed using Heidelberg Retinal Tomograph (HRT). Visual fields were analyzed with the Humphrey Visual Field analyzer. Statistical analyses were performed to discover the relationship between AH proteins and demographic characteristics, RNFL, optic nerve, and visual field parameters. AH reference database and website was developed using standard software tools including Visual Studio, ASP.NET, SQL, C#, and HTML. A total of 1774 unique proteins were identified in 251 AH samples of which 233 proteins were expressed in at least half of samples. Five protein families were discovered including apolipoproteins, complements, immunoglobulins, serine protease inhibitors (SERPINS) and insulin like growth factors (IGF). A total of 38 proteins significantly correlated with at least one RNFL thickness measure including average, inferior and superior thicknesses. Similarly, 62 proteins significantly associated with at least one HRT parameter such as cup shape measure, cup-disc area ratio and rim area. A total of 11, 9, 7, and 6 proteins were significantly correlated with pattern standard deviation, visual field index, mean deviation, and glaucoma hemifield test respectively. Strongly associated proteins include APOD, APOH, C4A, C4B, C7, IKHV3-9, IGKV2-28, SERPINA1, SERPINF1, IGFBP6, and IGFBP7. They are involved in immune responses, signaling, binding, and metabolic functions. These findings provide targets for future studies investigating molecular mechanisms and new therapies for glaucoma. Moreover, the database would serve as a resource for researchers pursuing AH proteomic and glaucoma studies.
    • MICROVASCULAR DYSFUNCTION IN HEART FAILURE WITH PRESERVED EJECTION FRACTION

      Davila, Alec Christopher; Biomedical Sciences (Augusta University, 2019-05)
      Background. Heart failure with preserved ejection fraction (HFpEF) is often manifested as impaired cardiac and microvascular reserve, for which no current effective therapies are available. We sought to determine if conducted vasodilation, which coordinates microvascular resistance longitudinally becomes compromised in HFpEF. We tested the hypothesis that inhibition of adenosine kinase (ADK), the major adenosine-metabolizing enzyme and novel therapeutic target, augments conducted vasodilation; therefore, improving tissue perfusion and left ventricle (LV) diastolic function. Methods and Results. Conducted vasodilation was assessed ex vivo in coronary arterioles isolated from right atrial appendages of patients with or without HFpEF diagnosis and in skeletal muscle arteries of the rodent model of HFpEF, ZSF1 rats. Obese ZSF1 rats displayed LV diastolic dysfunction over a 20-week lifespan as indicated by reduced E/A ratio and increased deceleration time of mitral flow velocity observed on echocardiogram. Conducted vasodilation in both HFpEF patients and obese ZSF1 rats were significantly reduced, which was associated with increased vascular expression of ADK. Isolated arterioles incubated with ADK inhibitor, ABT-702 (0.1 μM) displayed an improved conducted vasodilation. In vivo treatment of obese ZSF1 rats with ABT-702 (1.5 mg/kg, i.p. for 8-week) prevented LV diastolic dysfunction, and in a crossover design, ADK inhibition improved conducted vasodilation and LV diastolic function. Furthermore, ABT-702 treatment reduced surrogate markers of myocardial hypoxia (carbonic anhydrase 9 expression and fibrosis) in obese ZSF1 rats. Moreover, mice with endothelium-specific deletion of ADK exhibited augmented vasodilation and were protected against the development of transverse aortic constriction-induced LV dysfunction. Conclusion. Collectively, upregulation of microvascular ADK impairs conducted vasodilation in HFpEF. Pharmacological inhibition of ADK improves microvascular vasodilator function and provides beneficial effects on myocardial perfusion and LV diastolic function in HFpEF.
    • Assessment of Renal Ischemia Reperfusion Induced Injury in Male and Female Rats

      Crislip, Gene Ryan; Department of Physiology (2017)
      Acute kidney injury (AKI) is a clinical problem often induced by ischemia reperfusion (IR). Males are reported to have worse outcomes following IR compared to females based on measurements of blood urea nitrogen and creatinine. However, these markers are produced at different levels depending on body mass. The goal of Aims 1 and 2 was to do a complete assessment of the impact of sex on IR to establish a model that displays a sex difference. We measured multiple markers, including inulin clearance which is the gold standard of determining renal function. We determined there is no sex difference in response to IR after 24 hours. However, males had impaired renal function, higher vascular congestion and tubular injury than females 7 days following IR. A consequence of vascular congestion and tubular injury is fluid leakage into interstitial space, which increases renal volume. The goal of Aim 3 was to determine if ultrasound could be used as a tool to detect progressive changes in regional kidney volume following IR. To do this, we compared renal volume measurements with stereological assessment and examined the use of renal volume as an injury marker following IR. We verified the use of ultrasound to monitor renal volume after IR and the changes in volume correlated with the extent of medullary injury. Limiting vascular congestion improves recovery following IR. Pericytes are contractile cells that line the vessels in the renal medulla that are prone to congestion following IR. The goal of Aim 4 was to determine the role of renal pericytes following IR. To do this, we decreased pericytes in rats before IR to determine if this effected injury. We found that lower pericyte density was associated with greater vascular congestion following IR, additionally, males lose more pericytes than females. From these studies, we concluded that there was no sex difference in IR induced injury after 24 hours, however, following 7 days males had poorer recovery than females. We hypothesize that this poorer recovery is attributed to less pericytes in males following IR resulting in the inability to reduce vascular congestion compared to females.
    • Toll-like receptor 9 contributes to vascular dysfunction in hypertension

      McCarthy, Cameron; Department of Physiology (2016-03)
      Inappropriate immune system activation is common in hypertension; however, the exact mechanisms by which this occurs are not well understood. Innate immune system recognition and response to damage-associated molecular patterns (DAMPs) is becoming an increasingly accepted mechanism. Mitochondrial DNA (mtDNA) is a DAMP that is recognized by Toll-like receptor (TLR)9, and it is elevated in the circulation of spontaneously hypertensive rats (SHR). Therefore, we hypothesized that (1) inhibition of TLR9 in SHR with a TLR9 antagonist (ODN2088) or TLR9 inhibitor (chloroquine) would lower blood pressure and improve vascular function and that (2) treatment of normotensive rats with a TLR9 agonist (ODN2395) would cause vascular dysfunction and increase blood pressure. Both ODN2088 and chloroquine lowered high blood pressure in SHR and treatment with chloroquine also improved cyclooxygenase-dependent endothelial function and prevented the full recruitment of the adaptive immune system in SHR. On the other hand, treatment of normotensive rats with ODN2395 increased blood pressure and rendered their arteries less sensitive to acetylcholine-induced relaxation and more sensitive to norepinephrine-induced contraction. This dysfunctional vasoreactivity was due to cyclooxygenase activation, increased reactive oxygen species generation, and reduced nitric oxide bioavailability. In conclusion, these findings support the involvement of the innate immune system pattern recognition receptor TLR9 in the pathogenesis and maintenance of hypertension. Specifically, circulating mtDNA may activate TLR9 and contribute to high blood pressure and endothelial dysfunction in SHR.
    • Toll-like receptor 2 contributes to cerebrovascular dysfunction and cognitive impairment in diabetes

      Hardigan, Trevor; Department of Physiology (2016-03)
      The risk of cognitive decline in diabetes (Type 1 and Type 2) is significantly greater compared to normoglycemic patients, and the risk of developing dementia in diabetic patients is doubled. The etiology for this is likely multifactorial, but one mechanism that has gained increasing attention is decreased cerebral blood flow (CBF) as a result of cerebrovascular dysfunction. The innate immune system has been shown to play a role in diabetic vascular complications, notably through Toll-like receptor (TLR) stimulated release of proinflammatory cytokines and chemokines that leads to vascular damage. TLR2 has been implicated in the development of diabetic microvascular complications such as nephropathy, and thus we hypothesized that TLR2-mediated cerebrovascular dysfunction leads to decreased CBF and cognitive impairment in diabetes. Vascular TLR2 expression was increased and local TLR2 antagonism improved cerebrovascular function in diabetes. While the anti-hyperglycemic dipeptidylpeptidase-IV (DPP-IV) inhibitor linagliptin prevented TLR2 expression in brain microvascular endothelial cells (BMVEC) when applied locally, chronic in vivo treatment did not decrease vascular smooth muscle TLR2 expression. Treatment with linagliptin restored CBF in diabetes independent of effects on blood glucose levels, and this increase in CBF was correlated with decreased endothelin-1 (ET-1)-mediated vasoconstriction, decreased pathological remodeling, and increased endothelium-dependent relaxation. Knockout of TLR2 conferred protection from impaired CBF in early-stage diabetes and from hyperperfusion in long-term diabetes, prevented the development of endothelium dependent vascular dysfunction in diabetes, created a hyperactive and anxiolytic phenotype, and protected against diabetes induced impairment of long term hippocampal- and prefrontal cortex- mediated fear learning. In conclusion, these findings support the involvement of TLR2 in the pathogenesis of diabetic vascular disease and cognitive impairment.
    • Neuro-vascular Communication in the Hypothalamic Supraoptic Nucleus in Rats. Do nitric oxide and vasopressin play a role?

      Du, Wenting; Department of Physiology (2015-03)
      The classical model of neurovascular coupling (NVC) proposes that activity-dependent synaptically released glutamate dilates arterioles. However, whether this model is also applicable to brain areas that use less conventional neurotransmitters, such as neuropeptides, is currently unknown. To this end, we studied NVC in the hypothalamic magnocellular neurosecretory system (MNS) of the supraoptic nucleus (SON), in which dendritically released vasopressin (VP) can be found. Bath-applied VP significantly constricted SON arterioles via activation of the V ia receptor subtype. Vasoconstriction was also observed in response to single VP neuronal stimulation, an effect prevented by V ia receptor blockade (V2255). Conversely, osmotically-driven magnocellular neurosecretory neuronal population activity leads to a predominant nitric oxide (NO)- mediated vasodilation. Activity-dependent vasodilation was followed by a VP-mediated vasoconstriction, which acted to reset vascular tone. Taken together, our results unveiled a unique and complex form of NVC in the MNS, supporting a competitive balance between activity-dependent dendritic released VP and NO, in the generation of proper NVC responses.
    • The effects of hypertension on neurovascular unit function and structure

      Iddings, Jennifer Ann; Department of Physiology (2015)
      Functional hyperemia is the regional increase in cerebral blood flow upon increases in neuronal activity which ensures that the metabolic demands of the neurons are met. Hypertension is known to impair the hyperemic response; however, the neurovascular coupling mechanisms by which this cerebrovascular dysfunction occurs have yet to be fully elucidated. The goal of this dissertation project was to test the central hypothesis that hypertension-induced impairments in functional hyperemia are mediated by a specific disruption of communication within the neurovascular unit at the parenchymal arteriole level of the cerebrovascular tree. To test our hypothesis, we measured parenchymal arteriole reactivity, vascular smooth muscle cell Ca2+ dynamics, parenchymal arteriole remodeling and cerebral vascular density in cortical brain slices from normotensive (WKY) and hypertensive (SHR) rats. We found that vasoconstriction in response to the thromboxane A2 receptor agonist U46619 and basal vascular smooth muscle cell Ca2+ oscillation frequency were increased in parenchymal arterioles from SHR. In perfused and pressurized parenchymal arterioles, myogenic tone was increased in SHR. While K+-induced parenchymal arteriole dilations were similar in WKY and SHR, metabotropic glutamate receptor activation-induced parenchymal arteriole dilations were enhanced in SHR. Further, neuronal stimulation-evoked parenchymal arteriole dilations were similar in SHR and WKY. Parenchymal arteriole wall to lumen ratio and wall thickness were increased in SHR. Vascular density was also increased in deeper cortical layers in SHR. Our data indicate that although SHR parenchymal arterioles display vascular remodeling, neurovascular coupling is not impaired in SHR, at least at the parenchymal arteriole level.
    • The role of ceramide in the regulation of ciliogenesis

      He, Qian; Department of Physiology (2015)
      The primary cilium is a single, antenna-like protrusion of mammalian cells, involved in many signaling pathways important for cellular processes. In Madin-Darby Canine Kidney (MDCK) cells, an apical ceramide-enriched compartment (ACEC) was observed at the base of the primary cilia. Ceramide and Rab11a vesicles showed similar protein and lipid profiles. The lipid and protein composition suggested the presence of a ceramide associated lipid-protein complex containing atypical protein kinase C (aPKC), Cdc42, Sec8, Rab11a, and Rab8 in MDCK cells. Ceramide vesicles and Rab11a vesicles were highly enriched with C16 and C18 ceramides. Expression of a ceramide-binding but dominant-negative mutant of aPKC suppressed ciliogenesis, indicating that not only the association of ceramide with aPKC, but the activation of aPKC is critical for ciliogenesis in MDCK cells. In neural progenitors (NPs) differentiated from human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), the ceramide-protein interaction underlying ciliogenesis showed parallels to that with MDCK cells, but also significant differences. Ceramide was enriched in the apical region of the cell as well as primary cilia. In addition, the ceramide level was elevated by 3-fold after neural differentiation, especially C16 and C24:1 ceramide. Immunostaining showed that C16 ceramide was mainly distributed in the apical region and primary cilium, while the C24:1 antibody showed signals at the apicolateral cell membrane in addition to the apical area and the primary cilium. Immunostaining of aPKC also showed a signal at the apicolateral membrane as well as the primary cilium. Decreasing ceramide levels led to not only reduced ciliogenesis, but also translocation of membrane-bound aPKC to the cytosol, concurrent with its activation and the phosphorylation of its substrate Aurora A kinase (AurA). Incubation of ceramide-depleted cells with C24:1 ceramide restored ciliogenesis as well as membrane distribution of aPKC and accelerated neuronal process formation. The histone deacetylase (HDAC) inhibitor trichostatin A rescued ciliogenesis in ceramide-depleted MDCK cells and NPs, indicating that ceramide promotes tubulin acetylation in cilia. In summary, we concluded that ceramide promotes ciliogenesis by inhibiting HDAC6 activity in both of these two models, but via different molecular signaling pathways.
    • Integrated Effects of Leptin in the Forebrain and Hindbrain

      Desai, Bhavna N; Department of Physiology (2014-11)
      Obesity develops because of a sustained positive shift in energy balance. The hormone leptin was identified as a key negative feedback signal in energy balance regulation, yet it has been ineffective in reversing human obesity. Leptin injection studies in experimental animals have identified leptin receptors (ObRb) in the forebrain and hindbrain as critical and independent mediators of leptin responses. We hypothesized that under near physiological conditions; activation of ObRb in both these areas is required to reduce body fat. We used a male Sprague Dawley double cannulation rat model (3rd and 4th ventricle) and infused either saline (S) or sub-threshold doses of leptin (L) for 12 days (0.1μg leptin/24h in 3rd, 0.6μg leptin/24h in 4th) in different combinations SS, SL, LS, LL (3rd-4th), to test for integration of forebrain and hindbrain responses. There was no effect of leptin in single ventricle infused groups (LS, SL) compared to controls (SS). Rats with sub-threshold leptin infusions into both ventricles (LL) showed a 60% reduction in energy intake that reversed after day 6 and a 20% weight loss which stabilized at day 6. Body fat of LL rats was decreased by 30% in 6 days, and 50% after 12 days despite correction of energy intake. LL rats displayed normal activity and maintained normal energy expenditure despite weight loss. We further investigated which brain nuclei are involved in this integrated response using phosphorylation of signal transducer and activator of transcription 3 (pSTAT3) as a marker of ObRb activation and delta FosB (ΔFosB) as a marker of chronic neuronal activation. The weight loss in LL rats was associated with a significant increase in pSTAT3 and ΔFosB within multiple hypothalamic nuclei, including the arcuate, ventromedial and dorsomedial nuclei, with no changes in activation of brainstem nuclei. Our results suggest that under near physiologial conditions, the simultaneous activation of both forebain and hindbrain ObRb is required for leptin to reduce body fat and this is facilitated by leptin in the hindbrain promoting activation of pSTAT3 in the hypothalamus. This provides a new perspective on the physiological role of leptin and could lead to new strategies to treat obesity.
    • Genetic mutations cause primary aldosteronism

      Hattangady, Namita G; Department of Physiology (2014-10)
      The human adrenal glands are complex endocrine organs that are physiologically located above the kidney. The cortex of the adrenal gland may be considered as a combination of three different steroidogenic tissue-types which form concentric zones within each adrenal. The three cortical zones include zona glomerulosa (ZG), zona fasciculata (ZF) and zona reticularis (ZR). Each zone, under independent regulation, produces unique steroid(s) which exhibit specific functions. The outermost ZG layer secretes the steroid, aldosterone due to ZG specific expression of aldosterone synthase (CYP11B2). Aldosterone regulates sodium reabsorption, and therefore, blood pressure. Aldosterone production is tightly regulated by the renin-angiotensin-aldosterone system. Thus, aldosterone levels are in direct proportion with renin levels. Other known physiological regulators of aldosterone production include serum K+ and adrenocorticotrophic hormone. A type of endocrine hypertension termed ‘Primary Aldosteronism’ (PA), is characterized by aldosterone secretion under suppressed renin levels. PA accounts for almost 10 % of hypertension. More recently, genetic mutations in an inward rectifying K+ channel (KCNJ5) that occur as both, somatic and germline cases, have been implicated in the pathology of PA. The goal of this dissertation is to define the role of KCNJ5 mutations in PA. In this dissertation, I will summarize my studies that describe the acute and chronic events involved in mutated KCNJ5 mediated aldosterone excess. In addition, I will define a novel mutation in KCNJ5 of germline nature identified at Georgia Regents University. Finally, I will also describe some interesting lessons we learnt from the expression of mutated KCNJ5 in primary cultures of human adrenals. The prevalence of a hereditary form of PA termed as Familial Hyperaldosteronism type III (FH III) is very rare. Thus far, only a few mutations in the KCNJ5 gene, including T158A, G151R, G151E and I157S, are confirmed as causing FH III, following Mendelian genetics. Perhaps the most interesting feature of this disease is the varied phenotype between the different mutations. T158A-affected patients present with massive hyperplasia and require bilateral adrenalectomy. In contrast, patients affected by the G151E mutation have more severe hypertension, although their adrenals are near normal in appearance. In this study we identify a new germline mutation (Y152C). The index case was a 61 year old woman who underwent unilateral adrenalectomy. The patient with the Y152C mutation exhibited a milder hypertension phenotype (like the G151E-affected patient) with extensive hyperplasia (as seen in the T158A-affected patient). In vitro analyses of the Y152C mutation indicated a pathology similar to other known mutations in KCNJ5, including change in conductance to Na+ ions and elevated calcium levels, and increase in CYP11B2 mRNA and aldosterone production. The inherent challenge presented by current studies utilizing constitutive expression of KCNJ5 mutations is the limitation in studying acute temporal events such as post translational modifications of steroidogenic enzymes and transcription factors. To address this issue, we generated a doxycycline inducible cell model system for the T158A harboring KCNJ5 transgene. Herein, we demonstrate a useful system that was amenable to the study of acute and chronic events involved in mutant-KCNJ5 mediated aldosterone excess. Our findings suggest that mutant KNCJ5 increases CYP11B2 expression through the activation of transcriptional activators of CYP11B2. Additionally, this is the first study to demonstrate that mutant KCNJ5 also activates steroidogenic acute regulatory protein (StAR) at the levels of translation and post translational phosphorylation. We also demonstrate calcium channel blocker, verapamil as an efficient blocker of mKCNJ5 mediated aldosterone production. Finally, one of the sharp advantages of our study was the use of primary cultures of human adrenal cells to confirm the effects of mutated KCNJ5. Interestingly, transduction of cells with constitutive viruses for mutant KCNJ5, confirmed an increase in KCNJ5 mRNA, although no change in CYP11B2 expression levels was observed. Pilot data including treatment of primary cells with calcium ionophores indicated that ZF/ZR cells may have a phenotype that is ‘muted’ for calcium mediated pathways. We could also speculate that this may disprove some current hypotheses that APA harboring KCNJ5 mutations may originate from the ZF. Overall, this study has improved our knowledge regarding the pathogenesis of PA caused by KCNJ5 mutations and has identified verapamil as a potentially effective therapeutic strategy in the inhibition of aldosterone excess in this type of PA.
    • The Mechanism of Monomethylfumarate (MMF) as an Anti-psoriatic Agent

      Helwa, Inas; Department of Physiology (2014-09)
      Psoriasis is a chronic hyperproliferative inflammatory skin disorder whose primary etiology is not well understood. Keratinocytes play a pivotal role in the pathogenesis of psoriasis. The fumaric acid ester monomethylfuamarate (MMF) is the bioactive ingredient of the anti-psoriatic drug Fumaderm©, licensed in Germany since 1994. However, the exact mechanism of action of MMF is not yet well understood. Our data showed that MMF dose-dependently inhibited proliferation in primary murine and human keratinocytes and significantly increased the protein expression of the early marker of differentiation K10 and the activity of the late marker of differentiation transglutaminase enzyme. In addition, MMF inhibited mRNA expression of IL-6, TNFα and IL-1α and inhibited the protein expression of TNFα. Recently, the role of oxidative stress in psoriasis etiology has evolved and MMF has been shown to stimulate Nrf2 and mediate its nuclear translocation in other cell types. Therefore, we examined the effect of MMF on Nrf2 expression, localization and downstream effectors in keratinocytes. Nrf2 protein expression and nuclear translocation significantly increased following MMF treatment. Moreover, MMF significantly increased the mRNA expression of the Nrf2- downstream anti-oxidative enzymes, heme oxygense-1 and peroxiredoxin-6. MMF also decreased ROS generation in keratinocytes. Aquporin3 (AQP3) is a glycerol channel expressed in keratinocytes. Earlier studies from our group as well as others have shown that AQP3 plays a role in inducing early keratinocyte differentiation and that the activity of AQP3 correlates with its membranous localization. Therefore, we examined the effect of MMF on AQP3 expression and localization. MMF increased the mRNA and protein 3 expression of AQP3. In addition, MMF stimulated membranous translocation of AQP3 and increased glycerol uptake by keratinocytes. Eventually, we wanted to examine whether Nrf2 plays a role in the expression of AQP3. Our data showed that the Nrf2 stimulator sulforaphane (SFN) increased the expression of AQP3. Thus, our data suggest that MMF exerts its action through Nrf2 stimulation. Nrf2 stimulation helps to regain keratinocyte oxidative balance and may also play a role in inducing AQP3 expression and activity. This provides the molecular basis for the MMF-mediated improvement of keratinocyte differentiation and inhibition of keratinocyte proliferation.
    • The Role of Contralateral Cerebrovascular Myogenic Dysfunction in Stroke

      Coucha, Maha; Department of Physiology (2014-07)
      Acute ischemic stroke (AIS) is the fourth leading cause of death and disability in the United States. The only successful therapeutic target identified for the 800,000 annual victims of AIS is the cerebral vasculature. This emphasizes the importance of maintaining a well-functioning vasculature with a well-optimized myogenic tone to supply the necessary nutrients, and even the requisite concentration of neuroprotectant to the jeopardized tissue, but at the same time avoiding hemorrhage. While early studies described that ischemia/reperfusion (I/R) reduces cerebral perfusion in the nonischemic hemisphere as well, the underlying mechanisms and the impact of this contralateral vascular dysfunction on stroke outcomes have long been neglected. The goal of this proposal is to begin addressing this problem by focusing on the myogenic reactivity in ischemic and contralateral nonischemic hemispheres in experimental models with different stroke severity. Our global hypothesis is that contralateral myogenic dysfunction following I/R contributes significantly to stroke outcomes. This hypothesis will be tested by 1) determining the impact of I/R on myogenic reactivity in ischemic and contralateral hemispheres, and 2) determining the impact of contralateral myogenic dysfunction in conditions associated with poor stroke outcomes. This study will reveal the critical role of vascular dysfunction in nonischemic hemisphere in worsening stroke outcomes as well as the underlying mechanisms. The rationale is that once the mechanisms and modulators of cerebrovascular function and perfusion in both hemispheres are known, it will be possible to develop more effective strategies to deliver neuroprotective therapies to improve stroke outcomes and recovery. Moreover, these experiments have the potential to challenge the concept that contralateral hemisphere can serve as a control in preclinical stroke studies.
    • Mechanisms of ANG (1-7) Mediated Control of Blood Pressure in Males and Females

      Zimmerman, Margaret A.; Department of Physiology (2014-07)
      Angiotensin (Ang) (1-7) is a vasodilatory peptide of the renin angiotensin system (RAS). Ang (1-7) levels are greater in females, and Ang (1-7) blunts Ang II-mediated increases in blood pressure (BP) in females compared to males. The molecular mechanism(s) by which Ang (1-7) mediates BP regulation remains largely unknown, although Ang (1-7) has been suggested to increase nitric oxide (NO) levels, suppress proinflammatory markers, and contribute to the BP-lowering effects of RAS-inhibitors. The central hypothesis of my thesis is that Ang (1-7) contributes more to the molecular mechanisms that mediate BP control in females than males. To test this hypothesis, four aims were addressed. Aim 1 tested the hypothesis that the BP in male spontaneously hypertensive rats (SHR) is less sensitive to increases in Ang (1-7) than females. Ang (1-7) levels were pharmacologically increased in male and female SHR, and BP was assessed. However, Ang (1-7) infusion did not alter baseline BP in either sex. Aim 2 tested the hypothesis that Ang (1-7) contributes less to the BP-lowering effects of angiotensin receptor blockers (ARBs) in male than females SHR. To test this hypothesis, BP was measured in male and female SHR in response to an ARB. Males had the greater decrease in basal BP to an ARB than females, although female SHR were more sensitive to ARB-mediated inhibition of Ang II-induced increases in BP. Additional studies indicated that Ang (1-7) contributed to the BP-lowering effect of ARBs to a greater degree in females than in males. vi Aim 3 tested the hypothesis that Ang (1-7) contributes less to NO bioavailability in male than female SHR under basal conditions and following Ang II-hypertension. Ang (1-7) levels were pharmacologically increased or blocked in male and female SHR and the NO pathway was assessed. Renal cortical NO bioavailability was not affected by treatments in either sex. Finally, Aim 4 tested the hypothesis that Ang II infusion will increase renal T cells in both sexes; however, greater Ang (1-7) in females will result in more T regulatory cells (Tregs) relative to male Sprague Dawley (SD) rats. Renal T cells were increased in both males and females following chronic Ang II infusion, however, females exhibited an increase in immune-suppressive Tregs not seen in males. In contrast, males exhibited a greater increase in pro-inflammatory Th17 cells. Inhibition of Ang (1-7) did not alter the sex difference in Tregs, indicating that Ang (1-7) is not responsible for the greater increase in Tregs in females following Ang II-hypertension. In summary, this work examines the role of Ang (1-7) to mediate sex differences in BP regulation, where females are more dependent on Ang (1-7) than males to correct perturbations in the RAS.
    • Endothelin Receptor A Function in Progressive Kidney Disease

      Heimlich, Jonathan B.; Department of Physiology (2014-05)
      Endothelin-1 (ET-1) is a vasoactive peptide that regulates electrolyte and arterial blood pressure homeostasis. For this reason, ET-1 is increased under conditions of high dietary salt intake but also contributes to the progression of a variety of forms of kidney disease associated with hypoxia including sickle cell nephropathy. We previously found that ET-1 is an integral factor in glomerular injury seen during diabetic nephropathy through the activation of the ETA receptor. Recent studies also suggest ET-1 via the ETA receptor mediate the upregulation of reactive oxygen species (ROS) contributing to tissue damage. The overall aim of this work is to understand the role of ETA receptor activation in the development of sickle cell nephropathy and to determine the therapeutic potential of an ETA receptor antagonist in the treatment of renal manifestations associated with sickle cell disease (SCD). Since ET-1 is elevated and hypoxia is a prevalent occurrence in SCD, we utilized several strategies to elucidate the actions of ET-1 via the ETA receptor including ET-1 induction with a high salt diet and a hypoxic challenge to re-create an environment similar to SCD. The first aim was designed to determine whether ET-1 derived from endothelial cells contributes to oxidative stress in the glomerulus of mice subjected to a high salt diet and/or hypoxia. Hypoxia increased glomerular ET-1 mRNA expression in control, but not in vascular endothelial cell ET-1 knockout (VEET KO) mice. Increased superoxide formation was detected in the cortices of mice exposed to acute hypoxia. Under normoxic conditions, mice on a high salt diet had approximately 150% higher glomerular ET-1 mRNA expression compared to a normal salt diet. High salt diet administration also significantly increased glomerular ROS production in flox control, but not in glomeruli isolated from VEET KO mice. In C57BL6/J mice, the ETA receptor selective antagonist, ABT-627, significantly attenuated the increase in glomerular ROS production produced by high salt diet. In addition, chronic infusion of a sub-pressor dose of ET-1 via miniosmotic pumps in C57BL6/J mice significantly increased levels of glomerular ROS that were prevented by ETA antagonist treatment. Finally, three-hour exposure to hypoxia (8% O2) in control C57BL6/J mice significantly increased urinary protein excretion during the 24 hrs following hypoxia, but only in animals on a high salt diet. In conclusion, these data suggest that both hypoxia and a high salt diet increases glomerular ROS production via endothelial derived ET-1-ETA receptor activation and provide a potential mechanism for ET-1 induced nephropathy. The second aim was to test whether ET-1 acting via the ETA receptor contributes to renal injury in a mouse model of SCD. Humanized knockout, knockin sickle mice were used to describe the endothelin phenotype in the kidney. Sickle mice have increased ET-1 mRNA expression in both the cortex and glomeruli compared to heterozygous controls. Sickle animals have increased renal cortex ETA receptor mRNA expression but similar levels of ETB receptor mRNA expression. Radiolabeled ligand binding assays revealed sickle mice had increased ET-1 binding and subsequently increased ETA receptor binding in the renal vessels when compared to control mice. In response to PMA stimulation, sickle mice had increased glomerular ROS compared to controls, which could be prevented by treatment with ABT-627. Protein and nephrin excretion are two urinary markers of renal injury and were both found to be elevated in our mouse model of SCD. Treatment with ABT-627 resulted in significant decreases in both nephrin and protein excretion in SCD mice. Finally, 1 week of ETA antagonism also caused a significant decrease in mRNA expression of NADPH oxidase subunits as well as VEGF mRNA expression, both of which are thought to contribute to pathologic processes in chronic kidney disease. These data indicate a novel role for ET-1 in the progression of SCN, specifically via the ETA receptor in the glomerulus and suggest ETA antagonism to be a viable treatment strategy for SCN. Taken together, these studies reveal a prominent role for the pathologic activation of the ETA receptor in the progression of kidney diseases where ET-1 is upregulated and hypoxia is prevalent. ET-1 is known to contribute to other chronic kidney diseases such as diabetic nephropathy and significant benefit is found through ETA antagonism. These data offer a rationale for a novel treatment modality in renal disease associated with SCD.
    • The Mechanisms Underlying VLDL-Induced Aldosterone Production

      Tsai, Ying-Ying; Department of Physiology (2014-03)
      Aldosterone is responsible for sodium retention, thus increased blood volume and pressure. Excessive production of aldosterone results in high blood pressure, as well as renal disease, stroke, and visual loss via its effects on blood pressure. Although weight gain is associated with increased blood pressure, it remains unclear how excess fat deposits increase blood pressure. Indeed, overweight and obesity issues are correlated with serious health risks. In addition to hypertension, obese patients typically have high lipoprotein levels; moreover, some studies have suggested that aldosterone levels are also elevated and represent a link between obesity and hypertension. Very low density lipoprotein (VLDL) functions to transport triglycerides from the liver to peripheral tissues. Previous studies have demonstrated that VLDL can stimulate aldosterone production. By analogy with the signaling pathways activated by Angll, including the finding that VLDL increases cytosolic calcium levels, here we show that both phospholipase C (PLC) and phospholipase D (PLD) are involved in VLDL-induced aldosterone production. The effects of VLDL on steroidogeneses are mediated via an ability of these signaling pathways to result in the induction of steroidogenic acute regulatory (StAR) protein and aldosterone synthase (CYP11B2) expression, the early and late limiting steps in aldosterone biosynthesis, presumably byincreasing the phosphorylation (activation) of their regulatory transcription factors, such as the cAMP response element binding (CREB) protein family of transcription factors.