This collection contains theses and dissertations submitted by graduate students under the Department of Medicine for either a Master of Science degree or a Doctor of Philosophy degree.

Recent Submissions

  • 96 plays a role in the virulence of C. jejuni

    Rathbun, Kimberly M; Department of Medicine (2009-05)
    Campylobacter jejuni is a gastrointestinal pathogen of humans but part of the normal flora of poultry. C. jejuni therefore grows well at both 37°C and 42°C. Proteomic studies on temperature regulation in C. jejuni strain 81-176 revealed the upregulation at 37°C of CJ0596, a predicted periplasmic chaperone that is similar to proteins found to be involved in outer membrane protein (OMP) folding and virulence in other bacteria. The cj0596 gene was highly conserved in multiple strains and species of Campylobacter (24 in total), implying the importance of this gene. To study the role CJ0596 plays in Campylobacter pathogenesis, a mutant derivative of strain 81-176 was constructed in which the cj0596 gene was precisely deleted. This mutant was complemented by restoring the gene to its original chromosomal location. The mutant strain demonstrated a decreased growth rate and lower final growth yield, yet was more motile than wild-type. The cj0596 mutant also showed altered levels of several outer membrane proteins (OMPs), and changes in membrane-associated characteristics (antimicrobial sensitivity, autoagglutination, and biofilm formation). In either single or mixed infections, the mutant was less able to colonize mice than wild-type. Purified, recombinant CJ0596 had peptidyl-prolyl cistrans isomerase (PPIase) activitty, but did not functionally complement an E. coli surA mutant. These results suggest that C. jejuni CJ0596 is a PPIase and loss of CJ0596 alters phenotypes that have been shown to be related to the pathogenesis of the bacterium.
  • Mechanisms of ET-1-mediated 02~ production in the rat aorta

    Loomis, E. D.; Department of Medicine (2004-06)
    The objectives of this project were to test the hj^othesis that in the rat aorta endothelin-I (ET-I) binds to the ETa receptor stimulating superoxide (O2’ ) production. Furthermore, we wanted to identify the mechanism through which ET-1-mediates O2' production. Chemiluminescent detection of O2” production using probes such as lucigenin has been widely used with enzyme systems, leukocytes, and vascular tissues. Our first goal was to develop a microplate high-throughput protocol for lucigeninamplified chemiluminesence detection of 0 2 'L We have developed a novel adaptation to lucigenin-based assays that allows up to 36 samples to be counted at virtually the same time. Recent studies have shown that NOS 3 can become uncoupled and produce O2* when deprived of its cofactor BH4 . In addition several authors have shown that ONOOoxidizes BH4 in vitro. Using the high-throughput lucigenin assay and dihydroethidine (DHE) staining we have shown that (1) ET-1 is able to stimulate 0 2 "^ production in both endothelium-intact and -denuded vessels through the ETA-receptor, (2) ET-1 stimulates O2’ production through both NAD(P)H oxidase and an endothelial source of NOS, and (3) addition of exogenous tetrahydrobiopterin (BH4 ) and inhibition of peroxynitrite (ONOO-) inhibit ET-I-mediated 0 2 *^ production. Therefore our data have led us to hypothesize that ET-I stimulates O2* production by activating NAD(P)H oxidase through the ETa receptor. O2' production by NAD(P)H oxidase leads to the formation of ONOO- and the degradation of BH4. The loss of BH4 leads to uncoupled NOS which then contributes to ET-1-mediated production. In addition, we have found that (1) ET-1 increases the production of interleukin- 6 (IL-6 ) and (2) ET-1-mediated O2' production adversely affects vascular contractility. Although these consequences do not appear to be due to NOS uncoupling, they help support the role of ET-1 in vascular dysfunction.
  • Role of GCN2-dependent metabolic stress in regulating myeloid cell activation and differentiation.

    Liu, Haiyun; Department of Medicine (2014-12)
    Amino acid metabolism is a pivotal regulator of innate and adaptive immunity. During inflammation, myeloid cells expressing enzymes such as indoleamine 2, 3-dioxygenase (IDO) and arginase 1 (ARG1) that degrade the amino acids L-tryptophan (L-Trp) and L-arginine (L-Arg), respectively. This serves a critical role in controlling cellular survival, development, and function. Therefore, it is important to understand the metabolic stress sensing pathways that regulate immune cell behavior, which further control the overall inflammatory environment. General Control Non-depressible 2 (GCN2) is an integrated stress response (ISR) kinase activated by intracellular amino acid limitation. Activated GCN2 phosphorylates eukaryotic initiation factor 2 α (eIF2α), which leads to global translation repression while up-regulating various stress-associated transcription factors. We found that in a murine LPS-induced endotoxemia model, IDO expression in macrophages depleted L-Trp that activated the GCN2. GCN2 signaling promoted macrophage cytokine production (IL-6, IL-12), increased CHOP expression and NF-κB activation. GCN2 knockout (GCN2KO) mice showed significantly lower serum and splenic cytokine levels compared to wild-type (WT) mice, and were protected from septicemia induced mortality. In the murine EG7 tumor model, GCN2 signaling was also activated in myeloid-derived suppressor cells (MDSCs) mediated by ARG1 depletion of L-Arg. We found that GCN2 was required for transcription factor C/EBPβ induction and monocytic bone marrow MDSC (BM-MDSC) development. GCN2KO BM-MDSCs showed significantly reduced ARG1 activity and failed to suppress antigen-specific cytotoxic T lymphocytes (CTLs) in vitro and in vivo. GCN2KO mice also exhibited increased efficacy in eliminating tumor cells after adoptive CTL transfer therapy. These data suggest that myeloid cells actively deplete intracellular amino acids to regulate their own cellular behavior. Different amino acid metabolic stress signals converge on the GCN2 pathway which serves as a secondary messenger to modulate downstream transcription factors. Depending on the type of inflammation, GCN2 can either promote pro-inflammatory responses or the immunosuppressive function of myeloid cells. Thus, targeting the GCN2 pathway in myeloid cells may have great potential in clinical therapy.
  • Regulation of Virulence in the Human Pathogen Campylobacter jejuni by the RNA Binding Protein CsrA

    Fields, Joshua A; Department of Medicine (2011-10)
    Campylobacter jejuni is a leading bacterial cause of gastroenteritis in both the industrialized and developing world and has been associated with the onset of long term, debilitating sequelae such as Guillain-Barre Syndrome and reactive arthritis. The RNA binding protein CsrA (carbon storage regulator A), one of the relatively few regulatory elements in the C. jejuni genome, has been shown to regulate a number of processes in several other bacterial species including metabolism and virulence characteristics. We proposed the hypothesis that CsrA globally regulates C. jejuni pathogenesis via post-transcriptional repression or activation of virulence associated proteins. We created a csrA mutant in the C. jejuni strain 81-176 to investigate the role of CsrA in the virulence and physiology of the organism. In the absence of CsrA, we found that C. jejuni was no longer able to resist oxidative stresses, form biofilms, or adhere to intestinal epithelial cells in vitro in comparison to the wild type. We also found that C. jejuni was less motile than its parent strain and was defective in autoagglutination and fibronectin binding in vitro and mouse colonization in vivo. When we compared the proteome of the mutant strain to that of the wild type, we found that CsrA acted mostly upon the expression of proteins in stationary phase. In the absence of CsrA proteins responsible for various steps in C. jejuni metabolism, motility, oxidative stress responses, and epithelial cell adherence were differentially expressed. Finally, to further understand the molecular mechanisms of C. jejuni CsrA, we expressed it in a csrA mutant strain of E. coli. By heterologously expressing the C. jejuni protein in strain in which CsrA had been thoroughly characterized, we were able to show by complementation that C. jejuni CsrA was capable of both activating and repressing known targets of E. coli CsrA indicating that the molecular mechanisms of the two proteins are inherently the same.
  • Role of Oxidative Stress in High Endothelin Models of Hypertension

    Elmarakby, Ahmed A.; Department of Medicine (2004-07)
    Recent studies have shown that the potent vasoconstrictor peptide endothelin-1 (ET-1) stimulates superoxide production in vivo and in vitro. We hypothesized that ET-1 induced hypertension, at least in part, is due to an increase in oxidative stress. In the initial experiments, we hypothesized that ETA receptor stimulation contributes to the elevated blood pressure and superoxide production in ETB receptor deficient rats as an example of a high endothelin model of hypertension. Experiments were conducted on homozygous {si/si) ETB deficient and wild type {wt) rats fed a high salt diet for three weeks. Separate groups of rats were given normal drinking water or water containing the ETA receptor antagonist, ABT 627. On a normal salt diet, sl/sl rats had a significantly elevated systolic blood pressure (SBP) compared to wt. High salt caused a significant increase in SBP in sl/sl compared with wt rats. ETA receptor blockade decreased SBP in sl/sl rats on high salt without affecting the blood pressure in wt rats. Plasma 8-isoprostane levels, an indirect measure of oxidative stress, were significantly higher in sl/sl rats compared with the wt. ETa receptor blockade significantly attenuated the elevation in plasma 8- isoprostane levels in sl/sl rats'. These findings suggest that ET-1, through the ETA receptor, contributes to salt-induced hypertension and superoxide production in ETB deficient rats. We hypothesized that ET-1 increases superoxide production via the stimulation of the NADPH oxidase system. Chronic ET-1 infused rats were fed a high salt diet and either allowed to drink tap water, water containing the SOD mimetic, tempol, or the NADPH oxidase inhibitor, apocynin, for two weeks. Infusion of ET-1 increased mean arterial pressure (MAP) when compared to baseline values. Neither tempol nor apocynin treatment had any effect on the increase in MAP produced by ET 1. Plasma 8-isoprostane was increased significantly in ET-1 infused rats compared to rats on a high salt diet alone. Both tempol and apocynin treatment significantly attenuated the ET-1 induced increase in plasma 8-isoprostane. These data provide evidence that chronic ET-1 infusion increases vascular NADPH oxidase dependent superoxide production, but does not account for chronic ET-1-induced hypertension. Finally, experiments were performed to determine if increased kinins and/or decreased superoxide attenuates the elevation in blood pressure in chronic Ang II hypertensive rats. Four groups of rats, all given Ang II, were studied and allowed to drink tap water, water containing enalapril, tempol, or both for two weeks. Ang II infusion significantly increased SBP when compared with the baseline. Neither enalapril nor tempol treatment alone was able to attenuate the elevation in SBP. Combined administration of tempol and enalapril prevented the increase in SBP. Plasma 8-isoprostane was elevated significantly in Ang II infused rats when compared with control untreated rats. Tempol treatment alone or tempol plus enalapril significantly attenuated the increase in plasma 8-isoprostane. These studies support the hypothesis that an antioxidant alone is not effective in preventing Ang II hypertension. However, administration of an ACE inhibitor with an antioxidant enhances antioxidant efficiency in preventing Ang II hypertension. Overall, these studies showed that ETA receptor stimulation participates in superoxide production via the stimulation of NADPH oxidase and that antioxidant treatment alone is not sufficient to lower blood pressure in high endothelin models of hypertension.
  • Nitric Oxide Synthase Regulation in Inner Medullary Collecting Duct Cells

    Cai, Zheqing; Department of Internal Medicine (2001-06)
    Nitric oxide (NO) is a key regulator of sodium and water excretion in the kidney. It has been shown that renal tubules contain abundant nitric oxide synthase (NOS); however, little is known about the regulation of NOS expression and NOS activity in renal tubular cells. In the renal medulla, collecting duct cells produce a high level of endothelin-1 (ET-1), express caveolin-1 and protein tyrosine kinases (PTKs), and under certain conditions are exposed to high flows, resulting in an increased shear stress. In the current study, we hypothesize that ET-1 regulates expression of NOS isoform(s) and NOS activity is modulated by caveolin-1, tyrosine phosphorylation and shear stress. Western blot analysis and immunofluorescent staining showed that all three NOS isoforms were shown to be present in inner medullary collecting duct (IMCD) cells, a mouse IMCD cell line. After the IMCD cells were treated with 50 nM ET-1, NOS 1 was significantly and specifically increased, but not NOS 2 and NOS 3 expression. ET-1 also increased phosphorylation of p42/p44 MAPK in the IMCD cells. Genistein, a protein tyrosine kinase inhibitor, and PD 98059, a Mekl inhibitor, reduced the effects of ET-1 on phosphorylation of p42/p44 MAPK and up-regulation of NOS 1; furthermore, the ETA receptor antagonist, A 127722, rather than the ETB receptor antagonist, A 192621. inhibited the ET-1 effects in a concentration-dependent manner. The IMCD cells also Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. express caveolin-1, but none of the NOS isoforms appear to be associated with caveolin-1 by co-immunoprecipitation experiments, suggesting that caveolin-1 does not regulate NOS activity in the IMCD cells. NOS I is regulated by tyrosine phosphorylation and is shown to be phosphorylated at basal conditions. The non-specific inhibition of protein tyrosine kinases with 100 pM erbstatin A significantly increased nitrite production in the IMCD cell media. The tyrosine phosphorylation of NOS 1 was reduced by erbstatin A, and enhanced by vanadate, a protein tyrosine phosphatase inhibitor. When the IMCD cells were exposed to three levels of shear stress, 30, 10, 3.3 dyn/cnr for 1 hour, a significant increase in nitrite production was detected. L-NAME, a non-specific NOS inhibitor, completely blocked the effect of shear stress on nitrite production in IMCD cells. Therefore, in IMCD cells, NOS1 expression is up-regulated by ET-1 through activation of the ETA receptor and p42/p44 MAPK pathway; NO production is stimulated by tyrosine dephosphorylation, and activated by shear stress, but does not appear to be regulated by caveolin-1.
  • Mechanisms of Diet-Induced Hypertension and Vascular Disease Risk in Dahl Rats

    Spradley, Frank T.; Department of Medicine (2011)
    Dahl salt-sensitive (SS) rats are genetically predisposed to cardiovascular-renal disease. These studies examined cardiovascular-renal outcomes in response to a high-fat diet/normal-salt diet in SS rats. In a separate study, we examined cardiovascular-renal disease risk in SS rats on different standard chow diet/normal-salt diets. We tested the hypotheses that: (1) a high-fat diet induces hypertension and renal injury in SS rats; (2) a high-fat diet enhances aortic vasoconstriction in SS rats; (3) a high-fat diet induces aortic perivascular adipose tissue (PVAT) dysfunction in SS rats; and (4) two standard chow diets, namely AIN-76A and Teklad diet, induce differential vasoconstriction or vasorelaxation phenotypes in aorta and small mesenteric arteries from SS rats. In the high-fat diet studies, rats were provided high-fat diet starting at 12 weeks old. At 16 weeks old, SS rats on the high-fat diet had hypertension and greater renal glomerular and tubular injury than SS-13BN rats. SS rats supplemented with the immunosuppressive drug mycophenolate mofetil (MMF; 30 mg/kg/day, oral) for the duration of the high-fat diet did not develop hypertension. High-fat diet was associated with reduced vasoconstrictive response to angiotensin II and increased acetylcholine-mediated vasorelaxation in SS rats via increased nitric oxide synthase (NOS) function in comparison to SS rats maintained on a normal-fat/normal-salt diet. In regards to PVAT function, high-fat diet increased thoracic aorta PVAT deposition and induced PVAT-mediated blunting of aortic vasoconstriction. In the standard chow diet studies, 16-week old SS rats placed on the AIN or Teklad diet at weaning had similar NOS functional regulation of vasoconstriction and vasorelaxation in large and small arteries. However, by using a diet-switch protocol, we demonstrated that SS rats placed on AIN diet at weaning and changed to Teklad diet at 12 weeks old had reduced NOS-mediated vasorelaxation and reduced NOS buffering of vasoconstriction in small arteries from SS rats, which was not observed in the corresponding diet-switch group. In conclusion, these studies highlight differential renal and vascular responses to a short-term high-fat diet, and even changes in standard chow diet, when genetically predisposed to hypertension.
  • Role of Endothelin-1 (ET-1) in Glomerular Inflammation and Glomerular Permeability in Normal and Diabetic Kidneys

    Saleh, Mohamed Ahmed; Department of Medicine (2010-11)
    Endothelin-1 (ET-1) is a potent vasoactive peptide implicated in the pathogenesis of hypertension and renal disease. The overall specific aim of this dissertation is to investigate the role of ET-1 in mediating glomerular inflammation and permeability, especially in diseases characterized by high activity of the ET-1 system, such as diabetic nephropathy. The first study was designed to test the hypotheses that ET-1 increases albumin permeability of glomeruli isolated from normal rats and that chronic ET-1 infusion will increase glomerular permeability and inflammation independent of blood pressure. Glomerular permeability to albumin (Palb) was determined from the change in glomerular volume induced by exposing isolated glomeruli to oncotic gradients. Incubation of glomeruli taken from normal rats with ET-1 at a concentration that did not produce direct glomerular contraction (1 nM) significantly increased Palb, reaching a maximum after 4 hrs. Chronic ET-1 infusion for 2 weeks in Sprague-Dawley (SD) rats significantly increased Palb and nephrin excretion rate, effects that were attenuated in rats given an ETA receptor antagonist, ABT-627. Urinary protein and albumin excretion and mean arterial pressure (telemetry) were not changed by ET-1 infusion. Acute incubation of glomeruli isolated from ET-1-infused rats with the selective ETA antagonist significantly reduced Palb, an effect not observed with acute treatment with a selective ETB antagonist. Chronic ET-1 infusion increased glomerular and plasma sICAM-1 and MCP-1 and elevated the number of macrophages and lymphocytes in renal cortices (CD68- and CD3-positive staining, respectively). These effects were all attenuated in rats given an ETA selective antagonist. These data support the hypothesis that ET-1 directly increases glomerular permeability to albumin and renal inflammation via ETA receptor activation independent of changes in arterial pressure. The second study was designed to test the hypothesis that ETA receptor activation increases Palb and elevates pro-inflammatory markers in hyperglycemic rats. Male SD rats were given streptozotocin (STZ) or saline (sham). Half of the animals in each group received ABT- 627 beginning immediately after hyperglycemia had been confirmed. Glomeruli were isolated by sieving and Palb determined from the change in glomerular volume induced by exposing glomeruli to oncotic gradients of albumin. Glomerular nephrin expression was assessed by immunofluorescence, whereas urinary nephrin was measured by enzymelinked immunosorbent assay. Three and 6 weeks after STZ injection, proteinuria was significantly increased compared to sham controls and was significantly reduced by ABT-627 treatment. Palb was also increased at 3 and 6 wk post-STZ; ABT-627 had no effect on Palb or protein excretion in sham rats. Glomerular and plasma content of sICAM-1 and MCP-1 were significantly increased 6 wk after STZ. ABT-627 attenuated these increases. After 6 weeks of hyperglycemia, glomerular nephrin expression was decreased with a concurrent increase in urinary nephrin excretion; ABT-627 prevented glomerular nephrin loss in the hyperglycemic rats. These observations support the hypothesis that ET-1, via the ETA receptor, mediates the increase in proteinuria and Palb, possibly via nephrin loss, as well as early inflammation in the hyperglycemic rat. In the third study, we determined the actions of ETA and ETB receptors on measures of glomerular function and renal inflammation in the early stages of diabetic renal injury in rats. Six weeks after STZ-induced hyperglycemia, rats were given ABT-627 (5 mg/kg/d) a selective ETA antagonist; A-182086 (10 mg/kg/d), a combined ETA/B antagonist; or vehicle for 1 week. Sham controls received STZ vehicle (saline). Hyperglycemia led to significant proteinuria, increased Palb, nephrinuria, and an increase in total matrix metalloprotease (MMPs) and transforming growth factor-beta 1 (TGF-β1) activities in glomeruli. Plasma and glomerular sICAM-1 and MCP-1 were elevated after 7 weeks of hyperglycemia. Daily administration of both ABT-627 and A-182086 for 1 week significantly attenuated proteinuria, the increase in Palb, nephrinuria, and total MMPs and TGF-β1 activity. However, glomerular sICAM-1 and MCP-1 expression was attenuated with ABT-627, but not A-182086 treatment. In summary, both selective ETA and combined ETA/B antagonists reduced proteinuria, glomerular permeability and restored glomerular filtration barrier components integrity, but only ETA selective blockade had anti-inflammatory and anti-fibrotic effects. We conclude that selective ETA antagonists are more likely to be preferred for treatment of diabetic kidney disease.
  • The Role of Myostatin (GDF-8) in Chondrogenesis and Fracture Healing

    Elkasrawy, Moataz N.; Department of Medicine (2010-11)
    Traumatic musculoskeletal injuries frequently include damage to both muscle and bone where muscle injury itself can delay bone healing. Myostatin (GDF-8) is a member of the transforming growth factor-β (TGF-β) superfamily, and a negative regulator of skeletal muscle growth. Loss of myostatin function leads to a doubling of skeletal muscle mass, a general increase in bone density, and an increase in fracture callus bone volume. Myostatin is highly expressed during the first twenty-four hours after fracture, yet nothing is known about its role in fracture repair. We hypothesize that myostatin is a key regulator in the process of bone regeneration, and is a major therapeutic target for enhancement of fracture healing. Pharmacological inhibition of myostatin may therefore improve the regenerative capacity of both muscle and bone.
  • The Role of MHC-II Dependent Events in the Suppression Mediated by CD4+Foxp3+ Regulatory T cells

    Mmanywa, Faith Daima; Department of Medicine (2008-08)
    CD4+Foxp3+ regulatory T cells (Tregs) and antigen presenting cells (APCs) play an important role in maintaining peripheral tolerance but are otherwise exploited by tumors to create a state of unresponsiveness towards tumor antigens. The mechanisms of Treg mediated suppression are still not well understood. This work seeks to elucidate the role of major histocompatibility complex class II (MHC-II) dependent events in CD4+Foxp3+ Treg mediated suppression. The studies described here take advantage of novel conditional MHC-II deficient mice, which lack expression of MHC-II on peripheral APCs but still maintain their own naïve CD4 T cells and Tregs. In an in vitro system antigen-specific Tregs suppress CD8 T cell proliferation and effector molecule production in an antigen-specific and MHC-II dependent manner. In vivo, MHC-II deficiency resulted in a delay in tumor progression that was CD8 T cell dependent. We further describe two in vivo models in which the role of MHC-II dependent events in Treg mediated suppression can be tested. Therefore, a better understanding of Treg mediated suppression in the context of tumor-induced tolerance could provide potential strategies that could be utilized for anti-tumor immunotherapy.
  • The Role of Trigger Factor in Campylobacter Jejuni Pathogenesis

    Agee, Willie Andrew III; Department of Medicine; Georgia Regents University (2010-04)
    The first description of Campylobacter was by Theodor Esherich in 1886 when he published several articles noting a spiral bacterium in the colons of children who died of what Esherich called “cholera infantum”(10, 99) Esherich attempted to culture these spiral bacteria but was unable to do so (10, 99). He also found the bacteria in the stool of a significant number of infants suffering from gastrointestinal illness but he believed they played no role in the disease (10, 99). Unfortunately, because Esherich’s observations were published in German, his contribution in recognizing Campylobacter was not fully recognized until 1985 (10). Campylobacter was also recognized as a veterinary disease agent that causes abortion in ewes, sterility in cows and dysentery in calves (10). In 1909 veterinarians McFadyean and Stockman isolated a spiral bacterium from aborted sheep fetuses (10, 75). Ten years later Smith investigated cases of bovine abortion and isolated a spiral shaped bacterium (10, 98). He concluded that it was the same bacterium isolated by McFadyean and Stockman and proposed the name Vibrio fetus (10, 98). In 1949 Stegenga et al. discovered that V. fetus venerealis caused sterility in cows, and Florent showed there were two subspecies of V. fetus based on their biochemical and pathogenic characteristics (10, 23, 102). These subspecies were named V. fetus venerealis and V. fetus intestinalis based on causing sterility or dysentery respectively (10).
  • Regulation of renal medullary endothelin B receptor function by angiotensin II: evidence of sex differences

    Kittikulsuth, Wararat; Department of Medicine (2012-08)
    The renin angiotensin system and endothelin (ET) systems play critical roles in regulating kidney function and blood pressure. Angiotensin (Ang) II exerts its prohypertensive effects through AT1 receptor activation. ET-1 has similar effects mediated by ETA receptor stimulation. In contrast, ET-1, via ETB receptors, mediates vasodilation, anti-inflammation, and natriuresis. In the clinical setting, hypertension is more common in men than in premenopausal women of the same age. Moreover, in a number of animal models of genetic or experimental hypertension, females are somewhat protected from high blood pressure compared to males. We previously found that hypertensive male rats, induced by chronic Ang II infusion, have impaired ETB receptor function. Because ETB receptors are highly expressed in the renal medulla, the overall aim of this dissertation is to determine the role of Ang II in mediating renal medullary ETB receptor function, and to determine if differences in renal medullary ETB receptor function contribute to the sex differences observed in Ang II hypertension. The first aim was to test the hypothesis that renal medullary ETB receptor function is impaired in male Ang II hypertensive rats. However, ET-mediated natriuresis is preserved in female rats in response to chronic Ang II infusion. We compared the diuretic and natriuretic responses to intramedullary infusion of the ETB receptor agonist, sarafotoxin 6c (S6c), in male and female rats treated with Ang II (260 ng/kg/min s.c.) or vehicle for 14 days. Male Ang II hypertensive rats had impaired ETB-dependent sodium and water excretion. In contrast, renal medullary ETB receptor function was preserved in female Ang II-treated rats. Moreover, ETA-mediated diuretic and natriuretic responses were maintained in female Ang II hypertensive rats. These data demonstrate that, in contrast to male Ang II hypertensive rats, ET receptor-induced diuretic and natriuretic responses are preserved in female rats during chronic Ang II infusion. The second aim was to determine if ETB receptors limit the hypertensive response and renal injury induced by chronic Ang II infusion in female rats compared to males. Male and female rats received Ang II infusion (150 ng/kg/min; sc.) along with a high salt diet (4% Na) for 4 weeks; blood pressure was measured by telemetry. After one week of Ang II infusion with a high salt diet, subsets of both male and female rats received the ETB antagonist, A-192621, at three doses on consecutive weeks (1, 3, and 10 mg/kg/d in food). Male rats had significantly higher blood pressure compared to females after 4 weeks of Ang II. A-192621 resulted in a dose-dependent increase in blood pressure in female Ang II hypertensive rat while there was no significant change in males. After 4 weeks of Ang II infusion, the levels of proteinuria and nephrinuria were higher in male rats compared to female. A-192621 did not further increase urinary excretion of protein or nephrin in either male or female Ang II hypertensive rats. In conclusion, ETB receptors provide more protection against hypertension during chronic Ang II infusion in female rats compared to male. The third aim was to determine the physiological role of Ang II in regulating renal ETB receptor function during salt deprivation, a model with high levels of endogenous Ang II. After 2 weeks of normal (0.4% Na) or low (0.01-0.02% Na) salt feeding, the activation of ETB receptors in the renal medulla increased urine flow and sodium excretion of rats on normal salt diet. While urinary ET-1 excretion was comparable between a normal and low salt diet, ETB-dependent diuresis and natriuresis in response to acute intramedullary infusion of S6c was reduced in the low salt treated rats. Chronic treatment with the AT1 receptor antagonist, candesartan, restored ETB-induced water and sodium excretion in rats fed low salt diet. These findings support the hypothesis that AT1 receptors regulate renal medullary ETB receptor function in a low salt diet model to conserve sodium. From these studies, we conclude that Ang II via the AT1 receptor attenuates renal medullary ETB receptor function resulting in sodium and water retention. During pathological situations, Ang II has a greater inhibitory effect on ETB receptor function in male rats compared to females, leading to a greater increase in blood pressure in response to chronic Ang II infusion.
  • Novel Nitric Oxide Synthase-Dependent Mechanism of Vasorelaxation in Small Arteries from Hypertensive Rats

    Kang, Kyu-Tae; Department of Medicine (2007-10)
    Endothelial dysfunction in hypertension is associated with impaired endothelium-dependent vasorelaxation, which is consistently observed in conduit vessels. However, the controversial observation of either impaired or intact vasorelaxation of small resistance arteries from hypertensive animals suggests that the mechanism(s) of endothelium-dependent vasorelaxation in small resistance arteries may be different from that observed in conduit vessels under hypertensive condition. Vasorelaxation in small resistance arteries is mediated via multiple pathways including nitric oxide synthase (NOS)-, cyclooxygenase (COX)-, and endothelium-derived hyperpolarizing factor (EDHF)-mediated pathway. Therefore, the overall goal of these studies was to determine the mechanism(s) involving vasorelaxation of small arteries from hypertensive rats. For these studies, normotensive (NORM), angiotensin II-infused (ANG), high salt (HS), ANG high salt (ANG/HS), placebo, and deoxycorticosterone acetate-salt (DOCA) rats were studied. The studies with pharmacological blockade of each pathway demonstrated that the NOS-dependent component was increased to maintain acetylcholine (ACh)-induced vasorelaxation in small mesenteric arteries from hypertensive rats. Furthermore, increased NOS-dependent pathway appears to compensate for the dysfunctional Ca2+-activated K+ channel-sensitive EDHF pathway in small mesenteric arteries from ANG compared to NORM. These results led us to design further experiments to test the hypothesis that both NO and H2O2 serve as NOS-dependent mediators to maintain vasorelaxation in small mesenteric arteries from hypertensive rats. In small arteries from ANG, ACh increased NOS-dependent cGMP production. ACh also increased NOS3 phosphorylation at Ser 633 and decreased phosphorylation at Thr 495. While, NOS3 phosphorylation at Ser 1177 was impaired in response to ACh in ANG, which was accompanied by reduced basal and a less extended ACh-stimulated cGMP production in ANG compared to NORM. To investigate the alteration of signal transduction pathways related to impaired NOS3 phosphorylation at Ser 1177 in response to ACh, Akt phosphorylation at Ser 473 and VASP phosphorylation at Ser 239 were tested. These pathways were not changed by ACh in the small mesenteric arteries from ANG. Our results indicate that the NO/cGMP signaling is present in response to ACh in small mesenteric arteries from ANG, however this signaling pathway-mediating vasorelaxation may be facilitated via neither Akt nor PKG. On the other hand, ACh stimulated L-NAME-sensitive H2O2 production in small mesenteric arteries from ANG, but not NORM. H2O2 induced vasorelaxation and catalase blunted ACh-mediated vasorelaxation in small mesenteric arteries from ANG. Reduced BH4/BH2 ratio was observed in small mesenteric arteries from ANG compared to NORM, which might be one of the mechanisms of NOS-mediated H2O2 production. Antioxidant enzyme capacity was also determined in small mesenteric arteries from ANG and NORM. Total superoxide dismutase (SOD) activity and protein expression of CuZn SOD and ecSOD were reduced in ANG compared to NORM, while Mn SOD expression was comparable between groups. Interestingly, both activity and expression of catalase were reduced in ANG compared to NORM, whereas GPx activity and expression were not changed. These results indicate that reduced catalase activity and expression may contribute to the augmentation of H2O2 in small mesenteric arteries from ANG, whereas reduced SOD does not greatly influence the H2O2 production in both basal and ACh-stimulated condition. In conclusion, the NOS pathway appears to be the primary endothelium-derived relaxing factor (EDRF) pathway in small mesenteric arteries from experimental animal models of hypertension. The increased dependence on the NOS pathway in ACh-induced vasorelaxation is mediated by both NOS-derived NO/cGMP signaling and NOS-mediated H2O2.