Katherine Fredrich

Renal ischemia reperfusion inhibits VEGF expression and induces ADAMTS-1, a novel VEGF inhibitor

Reductions in vascular density occur following acute ischemia-reperfusion (I/R) injury that may predispose the development of chronic kidney disease. The mechanisms mediating vascular loss are not clear but may relate to the lack of effective vascular repair responses. To determine the regulation of the VEGF/VEGFR pathway following I/R injury, male Sprague-Dawley rats were subjected to bilateral renal ischemia (45 min) and allowed to recover for 1, 3, 7, and 35 days. VEGF mRNA expression was repressed by greater than 50% of control values up to 3 days postischemia, while VEGF protein was repressed for up to 7 days postischemia. The renal mRNA expression of receptors was not altered postischemia; however, VEGFR1 (flt-1) protein was transiently reduced in kidney while soluble flt-1 was elevated in plasma at 7 days following injury. Microarray analysis of angiogenesis-related genes identified the enhanced expression of a number of genes, among these was ADAMTS-1 (a disintegrin and metalloproteinase with thrombospondin motif-1), a secreted VEGF inhibitor. The altered expression of ADAMTS-1 was confirmed using RT-PCR and Western blot analysis; immunofluorescence localized its expression to proximal tubules following I/R injury. Other genes identified using microarray included aminopeptidase N, Smad-1, and Id-3 and their localization was also examined using immunohistochemistry. In summary, the data indicate no clear pattern of anti-angiogenic gene expression following renal I/R injury. However, the studies do suggest an overall inhibition of the VEGF pathway during the early injury and repair phase of renal ischemia that may contribute to an overall reduction in renal microvascular density.

Transfer of the CYP4A region of chromosome 5 from Lewis to Dahl S rats attenuates renal injury

This study examined the effect of transfer of overlapping regions of chromosome 5 that includes (4A(+)) or excludes (4A(-)) the cytochrome P-450 4A (CYP4A) genes from the Lewis rat on the renal production of 20-hydroxyeicosatetraenoic acid (20-HETE) and the development of hypertension-induced renal disease in congenic strains of Dahl salt-sensitive (Dahl S) rats. The production of 20-HETE was higher in the outer medulla of 4A(+) than in Dahl S or 4A(-) rats. Mean arterial pressure (MAP) rose to 190 +/- 7 and 185 +/- 3 mmHg in Dahl S and 4A(-) rats fed a high-salt (HS) diet for 21 days but only to 150 +/- 5 mmHg in the 4A(+) strain. Protein excretion increased to 423 +/- 40 and 481 +/- 37 mg/day in Dahl S and 4A(-) rats vs. 125 +/- 15 mg/day in the 4A(+) strain. Baseline glomerular capillary pressure (Pgc) was lower in 4A(+) rats (38 +/- 1 mmHg) than in Dahl S rats (42 +/- 1 mmHg). Pgc increased to 50 +/- 1 mmHg in Dahl S rats fed a HS diet, whereas it remained unaltered in 4A(+) rats (39 +/- 1 mmHg). Baseline glomerular permeability to albumin (P(alb)) was lower in 4A(+) rats (0.19 +/- 0.05) than in Dahl S or 4A(-) rats (0.39 +/- 0.02). P(alb) rose to approximately 0.61 +/- 0.03 in 4A(-) and Dahl S rats fed a HS diet for 7 days, but it remained unaltered in the 4A(+) rats. The expression of transforming growth factor-beta2 was higher in glomeruli of Dahl S rats than in 4A(+) rats fed either a low-salt (LS) or HS diet. Chronic administration of a 20-HETE synthesis inhibitor (HET0016; 10 mg.kg(-1).day(-1) sc) reversed the fall in MAP and renoprotection seen in 4A(+) rats. These results indicate that the introgression of the CYP4A genes from Lewis rats into the Dahl S rats increases the renal formation of 20-HETE and attenuates the development of hypertension and renal disease.

Dahl Salt-Sensitive Rats Are Protected Against Vascular Defects Related to Diet-Induced Obesity

Obesity increases plasma renin activity and angiotensin II levels, leading to vascular damage, elevated blood pressure, diabetes mellitus, and renal damage. Because genetic deletion of crucial parts of the renin-angiotensin system protect against obesity-related cardiovascular defects, we hypothesized that Dahl salt-sensitive (SS) rats, a model of chronically low plasma renin activity and angiotensin II levels, would be protected against vascular defects during diet-induced obesity compared with SS.13BN consomic rats showing normal renin-angiotensin system regulation. We evaluated vascular function in middle cerebral arteries of SS or SS.13BN rats fed high-fat (45% kcal from fat) versus normal-fat diet for 15 to 20 weeks from weaning. Endothelium-dependent relaxation in response to acetylcholine (10−8 to 10−4 mol/L) was restored in middle cerebral arteries of high-fat SS rats versus normal-fat diet controls, whereas vasodilation to acetylcholine was dramatically reduced in high-fat SS 13BN rats versus normal-fat diet controls. These findings support the hypothesis that physiological levels of angiotensin II play an important role in maintaining normal vascular relaxation in cerebral arteries and suggest that the cerebral vasculature of the SS rat model is genetically protected against endothelial dysfunction in diet-induced obesity.

Intestinal alkaline phosphatase administration in newborns decreases systemic inflammatory cytokine expression in a neonatal necrotizing enterocolitis rat model

BACKGROUND Supplementation of intestinal alkaline phosphatase (IAP), an endogenous protein expressed in the intestines, decreases the severity of necrotizing enterocolitis (NEC)-associated intestinal injury and permeability. We hypothesized that IAP administration is protective in a dose-dependent manner of the inflammatory response in a neonatal rat model. MATERIALS AND METHODS Pre- and full-term newborn Sprague-Dawley rat pups were sacrificed on day of life 3. Control pups were vaginally delivered and dam fed. Preterm pups were delivered via cesarean section and exposed to intermittent hypoxia and formula feeds containing lipopolysaccharide (NEC) with and without IAP. Three different standardized doses were administered to a group of pups treated with 40, 4, and 0.4U/kg of bovine IAP (NEC+IAP40, IAP4, or IAP0.4U). Reverse transcription-real-time polymerase chain reaction (RT-PCR) for inducible nitric oxide synthase (iNOS) and tumor necrosis factor (TNF)-α on liver and lung tissues and serum cytokine analysis for interleukin (IL)-1β, IL-6, IL-10, and TNF-α were performed. Data were analyzed by Kruskal-Wallis and Mann-Whitney tests, expressed as mean±standard error of the mean and P≤0.05 considered significant. RESULTS Levels of cytokines IL-1β, IL-6, and TNF-α increased significantly in NEC versus control, returning to control levels with increasing doses of supplemental enteral IAP. Hepatic and pulmonary TNF-α and iNOS messenger ribonucleic acid expressions increased in NEC, and the remaining elevated despite IAP supplementation. CONCLUSIONS Proinflammatory cytokine expression is increased systemically with intestinal NEC injury. Administration of IAP significantly reduces systemic proinflammatory cytokine expression in a dose-dependent manner. Early supplemental enteral IAP may reduce NEC-related injury and be useful for reducing effects caused by a proinflammatory cascade.

Intestinal Alkaline Phosphatase Is Protective to the Preterm Rat Pup Intestine

BACKGROUND Necrotizing enterocolitis (NEC) is the most common surgical emergency in neonates, with a mortality rate between 10 and 50%. The onset of necrotizing enterocolitis is highly variable and associated with numerous risk factors. Prior research has shown that enteral supplementation with intestinal alkaline phosphatase (IAP) decreases the severity of NEC. The aim of this study is to investigate whether IAP is protective to the preterm intestine in the presence of formula feeding and in the absence of NEC. METHODS Preterm rat pups were fed formula with or without supplementation with IAP, and intestine was obtained on day of life 3 for analysis of IAP activity, mRNA expression of TNFα, IL-6 and iNOS and permeability and cytokine expression after LPS exposure. RESULTS There was no difference in the absolute and intestine specific alkaline phosphatase activity in both groups. Rat pups fed IAP had decreased mRNA expression of the inflammatory cytokines TNFα, IL-6 and iNOS. Pups supplemented with IAP had decreased permeability and inflammatory cytokine expression after exposure to LPS ex vivo when compared to formula fed controls. CONCLUSIONS Our results support that IAP is beneficial to preterm intestine and decreases intestinal injury and inflammation caused by LPS.

Enteral intestinal alkaline phosphatase administration in newborns decreases iNOS expression in a neonatal necrotizing enterocolitis rat model.

PURPOSE To determine if intestinal alkaline phosphatase (IAP) decreases intestinal injury resulting from experimentally induced necrotizing enterocolitis (NEC). We hypothesized that IAP administration prevents the initial development of NEC related intestinal inflammation. METHODS Pre- and full-term newborn Sprague-Dawley rat pups were sacrificed on day 1 of life. Pre-term pups were exposed to intermittent hypoxia and formula containing LPS to induce NEC. Select NEC pups were given 40, 4 or 0.4 units/kg of bovine IAP (NEC+IAP40u, IAP4u or IAP0.4u) enterally, once daily. Ileal sections were evaluated by real-time PCR (qRT-PCR) for IAP, iNOS, IL-1β, IL-6, and TNF-α mRNA and immunofluorescence for 3-nitrotyrosine (3-NT). RESULTS Experimentally induced NEC decreased IAP mRNA expression by 66% (p ≤ 0.001). IAP supplementation increased IAP mRNA expression to control. Supplemental enteral IAP decreased nitrosative stress as measured by iNOS mRNA expression and 3-NT staining in the NEC stressed pups (p ≤ 0.01), as well as decreased intestinal TNF-α mRNA expression. In addition, IAP decreased LSP translocation into the serum in the treated pups. CONCLUSIONS We conclude that enterally administered IAP prevents NEC-related intestinal injury and inflammation. Enteral IAP may prove a useful strategy in the prevention of NEC in preterm neonates.

Early Enteral Stressors in Newborns Increase Inflammatory Cytokine Expression in a Neonatal Necrotizing Enterocolitis Rat Model

INTRODUCTION Inflammation in the premature intestine is a key factor that leads to the development of necrotizing enterocolitis (NEC). Activation of nuclear factor kappa B (NF-κB) and subsequent inflammation increases the severity of NEC. The aim of this study was to investigate the early temporal expression of inflammatory markers and activation of NF-κB in a neonatal rat model of NEC. METHODS Pre- and full-term newborn Sprague-Dawley rat pups were sacrificed at birth, 1.5, 4, 8, and 24 hours after receiving their first feed. Control pups were vaginally delivered and mother fed; NEC was induced by a combination of gavage feeding formula, hypoxia, and enteral lipopolysaccharide (LPS); and formula fed pups were fed every 4 hours with infant formula. Ileal tissue was collected for immunohistochemistry, real-time polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay. Serum was collected for cytokine content. Fold change of expression of inducible nitric oxide synthase (iNOS), interleukin (IL)-1β, IL-6, tumor necrosis factor-α (TNF-α), IL-10, NF-κB p65, and IκBα used RT-PCR. Data were analyzed by paired two-tailed t test, expressed as mean ± standard error of the mean, and p ≤ 0.05 considered significant. RESULTS No histologic injury was evident in ileal sections. At 1.5 h, iNOS expression increased twofold over control in NEC pups (2.1 vs. 1.0, p ≤ 0.05) and remained elevated at 24 h (0.7 vs. 9.4, p ≤ 0.05). IL-1β and IL-6 reached a peak at 24 h in NEC tissue compared with control. IL-10 expression rose in NEC pups after 4 h of insult and remained elevated in formula and NEC stressed pups. Coincident with an increase in p65 translocation into the nucleus and a reduction of IκBα detected in the cytoplasm, increased transcription of IκBα occurs. CONCLUSION These findings suggest that NF-κB activation initiates inflammation early in the course of NEC resulting in increased proinflammatory protein expression, underscoring the importance of the inflammatory response in this NEC model, which precedes evidence of histological injury.

AT1 Receptors Prevent Salt-Induced Vascular Dysfunction in Isolated Middle Cerebral Arteries of 2 Kidney–1 Clip Hypertensive Rats

BACKGROUND Elevated blood pressure, elevated angiotensin II (ANG II), and ANG II suppression with high salt (HS) diet all contribute to vascular dysfunction. This study investigated the interplay of HS diet and vascular function in a high renin model of hypertension. METHODS Male Sprague-Dawley rats were subjected to 2 kidney-1 clip (2K1C) Goldblatt hypertension for 4 weeks and compared with sham-operated controls. RESULTS Middle cerebral arteries (MCA) of 2K1C rats and sham-operated controls fed normal salt (NS; 0.4% NaCl) diet dilated in response to acetylcholine (ACh) and reduced partial pressure of oxygen (PO2). Switching to HS (4% NaCl) diet for 3 days to reduce plasma renin activity (PRA) eliminated vasodilation to ACh and reduced PO2 in sham-operated controls, with no effect on vasodilation in 2K1C rats. AT1 receptor blockade (losartan, 20 mg/kg/day; 1 week) eliminated vasodilator responses to ACh and reduced PO2 in 2K1C rats fed NS or HS diet. ANG II infusion (5 ng/kg/min, intravenous) for 3 days to prevent salt-induced reductions in plasma ANG II restored vascular relaxation in MCA of sham-operated controls fed HS diet. Copper/zinc superoxide dismutase expression and total superoxide dismutase activity were significantly higher in arteries of 2K1C rats fed HS diet vs. sham-operated controls. CONCLUSIONS These results suggest that the sustained effects of elevated ANG II levels in 2K1C hypertension maintain endothelium-dependent vasodilatation via AT1 receptor-mediated preservation of antioxidant defense mechanisms despite significant elevations in blood pressure and salt-induced suppression of PRA.

Intestinal NADPH Oxidase 2 Activity Increases in a Neonatal Rat Model of Necrotizing Enterocolitis

Necrotizing enterocolitis (NEC) is a complication of prematurity. The etiology is unknown, but is related to enteral feeding, ischemia, infection, and inflammation. Reactive oxygen species production, most notably superoxide, increases in NEC. NADPH oxidase (NOX) generates superoxide, but its activity in NEC remains unknown. We hypothesize that NOX-derived superoxide production increases in NEC. Newborn Sprague-Dawley rats were divided into control, formula-fed, formula/LPS, formula/hypoxia, and NEC (formula, hypoxia, and LPS). Intestinal homogenates were analyzed for NADPH-dependent superoxide production. Changes in superoxide levels on days 0-4 were measured. Inhibitors for nitric oxide synthase (L-NAME) and NOX2 (GP91-ds-tat) were utilized. RT-PCR for eNOS, NOX1, GP91phox expression was performed. Immunofluorescence studies estimated the co-localization of p47phox and GP91phox in control and NEC animals on D1, D2, and D4. NEC pups generated more superoxide than controls on D4, while all other groups were unchanged. NADPH-dependent superoxide production was greater in NEC on days 0, 3, and 4. GP91-ds-tat decreased superoxide production in both groups, with greater inhibition in NEC. L-NAME did not alter superoxide production. Temporally, superoxide production varied minimally in controls. In NEC, superoxide generation was decreased on day 1, but increased on days 3-4. GP91phox expression was higher in NEC on days 2 and 4. NOX1 and eNOS expression were unchanged from controls. GP91phox and p47phox had minimal co-localization in all control samples and NEC samples on D1 and D2, but had increased co-localization on D4. In conclusion, this study proves that experimentally-induced NEC increases small intestinal NOX activity. All components of NEC model are necessary for increased NOX activity. NOX2 is the major source, especially as the disease progresses.

Amelioration of salt-induced vascular dysfunction in mesenteric arteries of Dahl salt-sensitive rats by missense mutation of extracellular superoxide dismutase

Superoxide dismutase (SOD) enzymes, including extracellular SOD (ecSOD), are important for scavenging superoxide radicals (O2(·-)) in the vasculature. This study investigated vascular control in rats [SS-Sod(3m1Mcwi) (ecSOD(E124D))] with a missense mutation that alters a single amino acid (E124D) of ecSOD that produces a malfunctioning protein in the salt-sensitive (Dahl SS) genetic background. We hypothesized that this mutation would exacerbate endothelial dysfunction due to elevated vascular O2(·-) levels in SS, even under normal salt (NS; 0.4% NaCl) conditions. Aortas of ecSOD(E124D) rats fed standard rodent chow showed enhanced sensitivity to phenylephrine and reduced relaxation to acetylcholine (ACh) vs. SS rats. Endothelium-dependent dilation to ACh was unaffected by the mutation in small mesenteric arteries of ecSOD(E124D) rats fed NS diet, and mesenteric arteries of ecSOD(E124D) rats were protected from endothelial dysfunction during short-term (3-5 days) high-salt (HS; 4% NaCl) diet. ACh-induced dilation of mesenteric arteries of ecSOD(E124D) rats and SS rats fed NS diet was inhibited by N(G)-nitro-l-arginine methyl ester and/or by H2O2 scavenging with polyethylene glycol-catalase at higher concentrations of ACh. Total SOD activity was significantly higher in ecSOD(E124D) rats vs. SS controls fed HS diet, most likely reflecting a compensatory response to loss of a functional ecSOD isoform. These findings indicate that, contrary to its effect in the aorta, this missense mutation of ecSOD in the SS rat genome has no negative effect on vascular function in small resistance arteries, but instead protects against salt-induced endothelial dysfunction, most likely via compensatory mechanisms involving an increase in total SOD activity.