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Objective: The dominant-negative mutation, P467L, in peroxisome proliferator-activated receptor-γ (PPARγ) affects adipose tissue distribution, insulin sensitivity, and blood pressure in heterozygous humans. We hypothesized that the equivalent mutation, PPARγ-P465L, in mice will worsen atherosclerosis., Methods and Results: Apolipoprotein E-null mice with and without PPARγ-P465L mutation were bred in 129S6 inbred genetic background. Mild hypertension and lipodystrophy of PPARγ-P465L persisted in the apolipoprotein E-null background. Glucose homeostasis was normal, but plasma adiponectin was significantly lower and resistin was higher in PPARγ-P465L mice. Plasma cholesterol and lipoprotein distribution were not different, but plasma triglycerides tended to be reduced. Surprisingly, there were no overall changes in the atherosclerotic plaque size or composition. PPARγ-P465L macrophages had a small decrease in CD36 mRNA and a small yet significant reduction in very-low-density lipoprotein uptake in culture. In unloaded apolipoprotein E-null macrophages with PPARγ-P465L, cholesterol uptake was reduced whereas apolipoprotein AI-mediated efflux was increased. However, when cells were cholesterol loaded in the presence of acetylated low-density lipoprotein, no genotype difference in uptake or efflux was apparent. A reduction of vascular cell adhesion molecule-1 expression in aorta suggests a relatively antiatherogenic vascular environment in mice with PPARγ-P465L., Conclusions: Small, competing pro- and antiatherogenic effects of PPARγ-P465L mutation result in unchanged plaque development in apolipoprotein E-deficient mice.

Objectives: Deep brain stimulation (DBS) is an effective modality of treating cardinal motor symptoms of several movement disorders such as Parkinson's disease, essential tremor, and dystonia. Although hardware-related complications of DBS have been reported, the cosmetic satisfaction and discomfort associated with infraclavicular subcutaneous implantation of the pulse generator has not been described. The authors adopted a technique of transaxillary subpectoral implantable pulse generator (IPG) placement and investigated the difference in the discomfort, cosmetic satisfaction, mean operation time for IPG implantation, and severity of postoperative pain between infraclavicular subcutaneous placement and transaxillary subpectoral implantation of IPG., Materials and Methods: 25 patients who underwent bilateral, infraclavicular subcutaneous IPG placement for DBS and 15 patients who had bilateral, transaxillary subpectoral IPG placement were investigated., Results: The differences in cosmetic satisfaction and discomfort between the two groups were significant. The cosmetic satisfaction was higher and discomfort was less in the subpectoral IPG implantation group (p = 0.002 and p = 0.000). However, more time was needed for IPG implantation, and the postoperative pain was more severe after subpectoral IPG implantation (p = 0.002 and p = 0.000). There was no difference in cosmetic satisfaction according to sex (p = 0.907). There was one transient intercostobrachial nerve injury in the subpectoral IPG implantation group and two infections which needed removal of one side of the DBS hardware in the infraclavicular IPG implantation group., Conclusions: These results demonstrated that subpectoral transaxillary IPG implantation can provide better cosmetic satisfaction in patients undergoing DBS, with less discomfort and morbidity related to erosion and infection., (© 2011 International Neuromodulation Society.)

Excess soluble fms-like tyrosine kinase 1 (sFlt-1) of vascular endothelial growth factor receptor 1 secreted from the placenta causes pre-eclampsia-like features by antagonizing vascular endothelial growth factor signaling, which can lead to reduced endothelial nitric oxide synthase (eNOS) activity; the effect of this concomitant decrease in eNOS activity is unknown. We tested whether the decrease in nitric oxide occurring in female mice lacking eNOS aggravates the pre-eclampsia-like phenotype induced by increased sFlt-1. Untreated eNOS-deficient female mice had higher BP than wild-type mice. Adenovirus-mediated overexpression of sFlt-1 increased systolic BP by approximately 27 mmHg and led to severe loss of fenestration of glomerular capillary endothelial cells in both eNOS-deficient and wild-type mice. However, only the eNOS-deficient sFlt-1 mice exhibited severe foot process effacement. Compared with wild-type sFlt-1 mice, eNOS-deficient sFlt-1 mice also showed markedly higher urinary albumin excretion (467±74 versus 174±23 μg/d), lower creatinine clearance (126±29 versus 452±63 μl/min), and more severe endotheliosis. Expression of preproendothelin-1 (ET-1) and its ET(A) receptor in the kidney was higher in eNOS-deficient sFlt-1 mice than in wild-type sFlt-1 mice. Furthermore, the selective ET(A) receptor antagonist ambrisentan attenuated the increases in BP and urinary albumin excretion and ameliorated endotheliosis in both wild-type and eNOS-deficient sFlt-1 mice. Ambrisentan improved creatinine clearance and podocyte effacement in eNOS-deficient sFlt-1 mice. In conclusion, reduced maternal eNOS/nitric oxide exacerbates the sFlt1-related pre-eclampsia-like phenotype through activation of the endothelin system.

Oxidative stress contributes to the pathogenesis of diabetic nephropathy. In mitochondria, lipoic acid synthase produces α-lipoic acid, an antioxidant and an essential cofactor in α-ketoacid dehydrogenase complexes, which participate in glucose oxidation and ATP generation. Administration of lipoic acid abrogates diabetic nephropathy in animal models, but whether lower production of endogenous lipoic acid promotes diabetic nephropathy is unknown. Here, we crossed mice heterozygous for lipoic acid synthase deficiency (Lias(+/-)) with Ins2(Akita/+) mice, a well characterized model of type 1 diabetes. Double mutant mice had more overt diabetic nephropathy, including microalbuminuria, glomerular basement thickening, mesangial matrix expansion, and hypertension, compared with Lias(+/+)Ins2(Akita/+) controls. We also identified proximal tubules as a major site for generation of superoxide anions during diabetic nephropathy. Mitochondria in proximal tubular cells were particularly sensitive to damage in diabetic mice with reduced lipoic acid production. These results suggest that lipoic acid synthase deficiency increases oxidative stress and accelerates the development of diabetic nephropathy.

Objective: We retrospectively investigated the long-term results of percutaneous radiofrequency thermocoagulation (RFT) using fluoroscopic image-guidance for treatment of trigeminal neuralgia., Methods: A total of 38 patients diagnosed and treated with RFT as an idiopathic trigeminal neuralgia were investigated. To minimize the risks related to conventional technique based on cutaneous landmarks, and to eliminate the need to frequent reposition of cannula, we adopted a technique of image-guided fluoroscopic cannulation of the foramen ovale. To minimize sensory complication following thermal lesion, our target response was a generation of a lesion with mild to moderate hypalgesia rather than dense hypalgesia., Results: The immediate pain-relief was achieved in all patients underwent RFT. With mean duration of follow-up of 38.2 months (range,12-72), 11 (28.9%) experienced recurrence of pain. The mean timing of recurrence was 26.1 months (range,12-46). A 42.7% recurrence rate was estimated by Kaplan-Meier analysis for the 38 patients at 46 months; 20.2% within 2 years, 29.1% within 3 years. In the long-term, 27 patients (71%) and 6 patients (15.8%) showed Barrow Neurological Institute (BNI) score I and BNI score II responses. 3 (7.9%) patients was assessed as BNI score III, 2 patients (5.3%) showed BNI score IV response. As a complication, troublesome dysesthesia occurred in 3 of 38 patients (7.9%), however, there was no permanent cranial nerve palsy or morbidity., Conclusion: These results indicates that RFT under fluoroscopic image-guided cannulation of foramen ovale is a safe, effective, and reliable means of treating trigeminal neuralgia.

Objective: We sought to develop a murine model to examine the antithrombotic and antiinflammatory functions of human thrombomodulin in vivo., Methods and Results: Knock-in mice that express human thrombomodulin from the murine thrombomodulin gene locus were generated. Compared with wild-type mice, human thrombomodulin knock-in mice exhibited decreased protein C activation in the aorta (P<0.01) and lung (P<0.001). Activation of endogenous protein C following infusion of thrombin was decreased by 90% in knock-in mice compared with wild-type mice (P<0.05). Carotid artery thrombosis induced by photochemical injury occurred more rapidly in knock-in mice (12±3 minutes) than in wild-type mice (31±6 minutes; P<0.05). No differences in serum cytokine levels were detected between knock-in and wild-type mice after injection of endotoxin. When crossed with apolipoprotein E-deficient mice and fed a Western diet, knock-in mice had a further decrease in protein C activation but did not exhibit increased atherosclerosis., Conclusion: Expression of human thrombomodulin in place of murine thrombomodulin produces viable mice with a prothrombotic phenotype but unaltered responses to systemic inflammatory or atherogenic stimuli. This humanized animal model will be useful for investigating the function of human thrombomodulin under pathophysiological conditions in vivo.

The case of a 62-year-old woman who developed squamous cell carcinoma of the bladder 16 years after a kidney transplant is reported here. After the transplant, immunosuppressive therapy was maintained with cyclosporin A (200 mg/day) and the patient's serum creatinine level was 0.9 mg/dL. She was diagnosed with squamous cell carcinoma of the bladder 16 years later and underwent radical cystectomy with an orthotopic ileal neobladder. The Studer technique was used and the afferent ileal loop was anastomosed to the graft ureter. The postoperative course was uneventful. At the 6-month follow-up visit, the patient showed no evidence of recurrence. Her serum creatinine level was 1.0 mg/dL. The patient was continent during the day and the night. This case shows that the construction of an orthotopic ileal neobladder after cystectomy is safe and feasible in kidney transplant recipients.

Objective: We investigated the differential roles of apolipoprotein E (apoE) isoforms in modulating diabetic dyslipidemia-a potential cause of the increased cardiovascular disease risk of patients with diabetes., Research Design and Methods: Diabetes was induced using streptozotocin (STZ) in human apoE3 (E3) or human apoE4 (E4) mice deficient in the LDL receptor (LDLR(-/-))., Results: Diabetic E3LDLR(-/-) and E4LDLR(-/-) mice have indistinguishable levels of plasma glucose and insulin. Despite this, diabetes increased VLDL triglycerides and LDL cholesterol in E4LDLR(-/-) mice twice as much as in E3LDLR(-/-) mice. Diabetic E4LDLR(-/-) mice had similar lipoprotein fractional catabolic rates compared with diabetic E3LDLR(-/-) mice but had larger hepatic fat stores and increased VLDL secretion. Diabetic E4LDLR(-/-) mice demonstrated a decreased reliance on lipid as an energy source based on indirect calorimetry. Lower phosphorylated acetyl-CoA carboxylase content and higher gene expression of fatty acid synthase in the liver indicated reduced fatty acid oxidation and increased fatty acid synthesis. E4LDLR(-/-) primary hepatocytes cultured in high glucose accumulated more intracellular lipid than E3LDLR(-/-) hepatocytes concomitant with a 60% reduction in fatty acid oxidation. Finally, the exaggerated dyslipidemia in diabetic E4LDLR(-/-) mice was accompanied by a dramatic increase in atherosclerosis., Conclusions: ApoE4 causes severe dyslipidemia and atherosclerosis independent of its interaction with LDLR in a model of STZ-induced diabetes. ApoE4-expressing livers have reduced fatty acid oxidation, which contributes to the accumulation of tissue and plasma lipids.

Sodium wasting during the neonatal period is the consequence of a physiological aldosterone resistance, related to a low renal mineralocorticoid receptor (MR) expression at birth, both in humans and mice. To investigate whether aldosterone is involved in the neonatal regulation of MR expression, we compared aldosterone and corticosterone levels and renal MR expression by quantitative real-time PCR, between aldosterone synthase (AS) knockout, heterozygous, and wild type (WT) mice, at birth and postnatal d 8. Analysis of MR transcripts showed a similar expression profile in all genotypes, demonstrating that the lack of aldosterone does not modify either the low renal MR expression at birth or its postnatal induction. However, mRNA levels of the α-subunit of the epithelial sodium channel, a MR target gene, were significantly higher in WT compared with AS knockout mice, both at birth and postnatal d 8, despite high corticosterone levels in AS knockout mice, indicating that aldosterone is required for optimal renal induction of the epithelial sodium channel. Using organotypic cultures of newborn WT kidneys, we confirmed that aldosterone does not regulate MR expression at birth, but is instead capable of increasing MR expression in mature kidneys, unlike dexamethasone. In sum, we demonstrate both in vivo and in vitro, that, whereas aldosterone has no significant impact on renal MR expression at birth, it is crucial for optimal MR regulation in postnatal kidneys and for appropriate hydroelectrolytic balance. Understanding of MR-regulatory mechanisms could therefore lead to new therapeutic strategies for the management of sodium loss in preterms and neonates.

Diabetes and the development of its complications have been associated with mitochondrial DNA (mtDNA) dysfunction, but causal relationships remain undetermined. With the objective of testing whether increased mtDNA mutations exacerbate the diabetic phenotype, we have compared mice heterozygous for the Akita diabetogenic mutation (Akita) with mice homozygous for the D257A mutation in mitochondrial DNA polymerase gamma (Polg) or with mice having both mutations (Polg-Akita). The Polg-D257A protein is defective in proofreading and increases mtDNA mutations. At 3 mo of age, the Polg-Akita and Akita male mice were equally hyperglycemic. Unexpectedly, as the Polg-Akita males aged to 9 mo, their diabetic symptoms decreased. Thus, their hyperglycemia, hyperphagia and urine output declined significantly. The decrease in their food intake was accompanied by increased plasma leptin and decreased plasma ghrelin, while hypothalamic expression of the orexic gene, neuropeptide Y, was lower and expression of the anorexic gene, proopiomelanocortin, was higher. Testis function progressively worsened with age in the double mutants, and plasma testosterone levels in 9-mo-old Polg-Akita males were significantly reduced compared with Akita males. The hyperglycemia and hyperphagia returned in aged Polg-Akita males after testosterone administration. Hyperglycemia-associated distal tubular damage in the kidney also returned, and Polg-D257A-associated proximal tubular damage was enhanced. The mild diabetes of female Akita mice was not affected by the Polg-D257A mutation. We conclude that reduced diabetic symptoms of aging Polg-Akita males results from appetite suppression triggered by decreased testosterone associated with damage to the Leydig cells of the testis.

Polymorphisms in the human endothelial nitric oxide synthase (eNOS) gene (NOS3) have been associated with advanced nephropathy in diabetic patients and with decreased expression in tissue culture. However, direct proof that modest genetic decreases in eNOS expression worsen diabetic nephropathy is lacking. To investigate this effect, we took advantage of the hybrid vigor and genetic uniformity of the F1 progeny (eNOS(+/+), eNOS(+/-), or eNOS(-/-) with or without diabetes) of a cross between heterozygous 129S6/SvEvTac eNOS(+/-) inbred females and heterozygous C57BL/6J eNOS(+/-) inbred males carrying the dominant Akita diabetogenic mutation Ins2(C96Y/+). Whereas all C57BL/6J inbred eNOS(-/-) and eNOS(+/-) diabetic mice died before 5 mo, almost half of the F1 hybrid eNOS(-/-) and eNOS(+/-) diabetic mice lived until killed at 7 mo. Heterozygous eNOS(+/-) diabetic mice expressed ∼35% eNOS mRNA in the kidney and ∼25% glomerular eNOS protein relative to their eNOS(+/+) diabetic littermates. These decreases in eNOS elevated blood pressure (BP) but not blood glucose. Urinary albumin excretion, mesangial expansion, glomerulosclerosis, mesangiolysis, and glomerular filtration rate increased in the order: eNOS(+/+) Akita < eNOS(+/-) Akita < eNOS(-/-) Akita, independently of BP. Glomerular basement membrane thickening depended on increased BP. Renal expression of tissue factor and other inflammatory factors increased with the nephropathy; Nos2 also increased. Surprisingly, however, decreased eNOS expression ameliorated the increases in oxidative stress and tubulointerstitial fibrosis caused by diabetes. Our data demonstrate that a modest decrease in eNOS, comparable to that associated with human NOS3 variants, is sufficient to enhance diabetic nephropathy independently of its effects on BP.

Background: Oxidative stress, an excessive production of reactive oxygen species (ROS) outstripping antioxidant defense mechanisms, occurs in cardiovascular pathologies, including hypertension. In the present study, we used biochemical, physiological, and pharmacological approaches to explore the role of derangements of catecholamines, ROS, and the endothelium-derived relaxing factor nitric oxide (NO(•)) in the development of a hyperadrenergic model of hereditary hypertension: targeted ablation (knockout [KO]) of chromogranin A (Chga) in the mouse., Methods and Results: Homozygous ⁻(/)⁻ Chga gene knockout (KO) mice were compared with wild-type (WT, +/+) control mice. In the KO mouse, elevations of systolic and diastolic blood pressure were accompanied by not only elevated catecholamine (norepinephrine and epinephrine) concentrations but also increased ROS (H₂O₂) and isoprostane (an index of lipid peroxidation), as well as depletion of NO(•). Renal transcript analyses implicated changes in Nox1/2, Xo/Xdh, and Sod1,2 mRNAs in ROS elevation by the KO state. KO alterations in blood pressure, catecholamines, H₂O₂, isoprostane, and NO(•) could be abrogated or even normalized (rescued) by either sympathetic outflow inhibition (with clonidine) or NADPH oxidase inhibition (with apocynin). In cultured renal podocytes, H₂O₂ production was substantially augmented by epinephrine (probably through β₂-adrenergic receptors) and modestly diminished by norepinephrine (probably through α₁-adrenergic receptors)., Conclusions: ROS appear to play a necessary role in the development of hyperadrenergic hypertension in this model, in a process mechanistically linking elevated blood pressure with catecholamine excess, renal transcriptional responses, ROS elevation, lipid peroxidation, and NO(•) depletion. Some of the changes appear to be dependent on transcription, whereas others are immediate. The cycle could be disrupted by inhibition of either sympathetic outflow or NADPH oxidase. Because common genetic variation at the human CHGA locus alters BP, the results have implications for antihypertensive treatment as well as prevention of target-organ consequences of the disease. The results document novel pathophysiological links between the adrenergic system and oxidative stress and suggest new strategies to probe the role and actions of ROS within this setting.

Connective tissue growth factor (CTGF) is a signaling molecule that primarily functions in extracellular matrix maintenance and repair. Increased Ctgf expression is associated with fibrosis in chronic organ injury. Studying the role of CTGF in fibrotic disease in vivo, however, has been hampered by perinatal lethality of the Ctgf null mice as well as the limited scope of previous mouse models of Ctgf overproduction. Here, we devised a new approach and engineered a single mutant mouse strain where the endogenous Ctgf-3' untranslated region (3'UTR) was replaced with a cassette containing two 3'UTR sequences arranged in tandem. The modified Ctgf allele uses a 3'UTR from the mouse FBJ osteosarcoma oncogene (c-Fos) and produces an unstable mRNA, resulting in 60% of normal Ctgf expression (Lo allele). Upon Cre-expression, excision of the c-Fos-3'UTR creates a transcript utilizing the more stable bovine growth hormone (bGH) 3'UTR, resulting in increased Ctgf expression (Hi allele). Using the Ctgf Lo and Hi mutants, and crosses to a Ctgf knockout or Cre-expressing mice, we have generated a series of strains with a 30-fold range of Ctgf expression. Mice with the lowest Ctgf expression, 30% of normal, appear healthy, while a global nine-fold overexpression of Ctgf causes abnormalities, including developmental delay and craniofacial defects, and embryonic death at E10-12. Overexpression of Ctgf by tamoxifen-inducible Cre in the postnatal life, on the other hand, is compatible with life. The Ctgf Lo-Hi mutant mice should prove useful in further understanding the function of CTGF in fibrotic diseases. Additionally, this method can be used for the production of mouse lines with quantitative variations in other genes, particularly with genes that are broadly expressed, have distinct functions in different tissues, or where altered gene expression is not compatible with normal development.

Study Design: A retrospective study of clinical results of operative treatment for degenerative lumbar scoliosis., Objective: To determine the risk factors of sagittal decompensation after long instrumentation and fusion to L5 or S1., Summary of Background Data: Little is known about the risk factors for sagittal decompensation, which was defined in this study as sagittal C7 plumb falling anterior >8 cm from the posterosuperior corner of the sacrum., Methods: Forty-five patients (mean age: 64.4 year) with adult degenerative lumbar scoliosis were reviewed retrospectively with a minimum 2 years. The mean number of levels fused was 6.1 +/- 1.6 segments. The upper instrumented vertebra ranged from T9 to L2. The lower instrumented vertebra was L5 and S1 in 24 and 21 patients, respectively., Results: Sagittal decompensation (SD) developed in 19 patients. The most significant risk factors of SD were preoperative sagittal imbalance and high pelvic incidence. The preoperative sagittal C7 plumb was more positive (67.9 mm) in the decompensation group than in the balance group (37.0 mm) (P = 0.002). There was a significant difference in pelvic incidence between 61.7 degrees in the decompensation and 54.9 degrees in the balance group (P = 0.01). The preoperative lumbar lordosis was hypolordotic in the decompensation group, however, it was not found to be a risk factor. Pseudarthrosis was identified at the lumbosacral junction in 5 patients, and 4 of them (80%) had SD. SD developed in 55% of patients who had loosening of the distal screws and 50% of patients with hypolordotic lumbar fusion. Distal adjacent segment disease was more likely to cause SD than proximal adjacent segment disease., Conclusion: Sagittal decompensation is common after long posterior instrumentation and fusion for degenerative lumbar scoliosis. It is mostly associated with complications at the distal segments, including pseudarthrosis and implant failure at the lumbosacral junction. Restoration of optimal lumbar lordosis and secure lumbosacral fixation is necessary especially in patients with preoperative sagittal imbalance and high pelvic incidence in order to prevent sagittal decompensation after surgery.

Activation of the immune system by ANG II contributes to the pathogenesis of hypertension, and pharmacological suppression of lymphocyte responses can ameliorate hypertensive end-organ damage. Therefore, to examine the mechanisms through which lymphocytes mediate blood pressure elevation, we studied ANG II-dependent hypertension in scid mice lacking lymphocyte responses and wild-type controls. Scid mice had a blunted hypertensive response to chronic ANG II infusion and accordingly developed less cardiac hypertrophy. Moreover, lymphocyte deficiency led to significant reductions in heart and kidney injury following 4 wk of angiotensin. The muted hypertensive response in the scid mice was associated with increased sodium excretion, urine volumes, and weight loss beginning on day 5 of angiotensin infusion. To explore the mechanisms underlying alterations in blood pressure and renal sodium handling, we measured gene expression for vasoactive mediators in the kidney after 4 wk of ANG II administration. Scid mice and controls had similar renal expression for interferon-gamma, interleukin-1beta, and interleukin-6. By contrast, lymphocyte deficiency (i.e., scid mice) during ANG II infusion led to upregulation of tumor necrosis factor-alpha, endothelial nitric oxide synthase (eNOS), and cyclooxygenase-2 (COX-2) in the kidney. In turn, this enhanced eNOS and COX-2 expression in the scid kidneys was associated with exaggerated renal generation of nitric oxide, prostaglandin E(2), and prostacyclin, all of which promote natriuresis. Thus, the absence of lymphocyte activity protects from hypertension by allowing blood pressure-induced sodium excretion, possibly via stimulation of eNOS- and COX-2-dependent pathways.

Mutations and polymorphisms in PPARG have been linked to adiposity and partial lipodystrophy in humans. However, how disturbances in PPARG lead to depot-specific effects on adipose tissue, as shown by the characteristic aberrant fat distribution in patients, remains unclear. By manipulating the 3'-untranslated region of the Pparg gene, we have generated mice with peroxisome proliferator-activated receptor gamma (PPAR gamma) gene expression ranging from 25% to 100% normal. Basal levels of PPAR gamma transcripts between 50% and approximately 100% had no significant effect on body weight, fat mass, and insulin sensitivity. In contrast, mice with 25% normal PPAR gamma expression exhibited reduced body weight and total fat mass, insulin resistance, and dyslipidemia. Interestingly, fat mass was selectively reduced in perigonadal depot without significant changes in inguinal and other depots. Expression of adipogenic factor CCAAT enhancer binding protein-alpha and some other metabolic genes containing peroxisome proliferator response element were reduced in a perigonadal depot-specific fashion. This was further associated with depot-specific reduction in the expression of adipokines, increased expression of TNFalpha, and increased ectopic lipid deposition in muscles. Together, these results underscore the differential sensitivity of the individual fat depots on PPAR gamma availability as an underlying mechanism of partial lipodystrophy.

To test the hypothesis that angiotensin (Ang) II induces profibrotic gene expression through endogenous aldosterone, we measured the effect of 4 h infusion (600 ng/kg x min) of Ang II on tissue mRNA expression of plasminogen activator inhibitor 1 (PAI-1), preproendothelin-1 (ppET-1), TGF-beta, and osteopontin in wild-type (WT), aldosterone synthase-deficient (AS(-/-)), and AS(-/-) mice treated with aldosterone (either 500 ng/d for 7 d or 250 ng as a concurrent 4 h infusion). Ang II increased aldosterone in WT (P < 0.001) but not in AS(-/-) mice. Aldosterone (7 d) normalized basal aldosterone concentrations in AS(-/-) mice; however, there was no further effect of Ang II on aldosterone (P = NS). Basal cardiac and aortic PAI-1 and ppET-1 expression were similar in WT and AS(-/-) mice. Ang II-stimulated PAI-1 (P < 0.001) and ppET-1 expression (P = 0.01) was diminished in the heart of AS(-/-) mice; treatment with aldosterone for 4 h or 7 d restored PAI-1 and ppET-1 mRNA responsiveness to Ang II in the heart. Ang II increased PAI-1 (P = 0.01) expression in the aorta of AS(-/-) as well as WT mice. In the kidney, basal PAI-1, ppET-1, and TGF-beta mRNA expression was increased in AS(-/-) compared with WT mice and correlated with plasma renin activity. Ang II did not stimulate osteopontin or TGF-beta expression in the heart or kidney. Endogenous aldosterone contributes to the acute stimulatory effect of Ang II on PAI-1 and ppET-1 mRNA expression in the heart; renin activity correlates with basal profibrotic gene expression in the kidney.

Activation of the renin-angiotensin system contributes to the progression of chronic kidney disease. Based on the known cellular effects of ANG II to promote inflammation, we posited that stimulation of lymphocyte responses by ANG II might contribute to the pathogenesis of hypertensive kidney injury. We therefore examined the effects of the immunosuppressive agent mycophenolate mofetil (MMF) on the course of hypertension and kidney disease induced by chronic infusion of ANG II in 129/SvEv mice. Although it had no effect on the severity of hypertension or cardiac hypertrophy, treatment with MMF significantly reduced albuminuria and ameliorated kidney injury, decreasing glomerulosclerosis and reducing lymphocyte infiltration into the renal interstitium. Attenuation of renal pathology with MMF was associated with reduced expression of mRNAs for the proinflammatory cytokines interferon-gamma and tumor necrosis factor-alpha and the profibrotic cytokine transforming growth factor-beta. As infiltration of the kidney by T lymphocytes was a prominent feature of ANG II-dependent renal injury, we carried out experiments examining the effects of ANG II on lymphocytes in vitro. We find that exposure of splenic lymphocytes to ANG II causes prominent rearrangements of the actin cytoskeleton. These actions require the activity of Rho kinase. Thus, ANG II exaggerates hypertensive kidney injury by stimulating lymphocyte responses. These proinflammatory actions of ANG II seem to have a proclivity for inducing kidney injury while having negligible actions in the pathogenesis of cardiac hypertrophy.

Reexpression of the fetally expressed beta-myosin heavy chain (beta-MHC) gene is a well documented marker of pathological cardiac hypertrophy and normal aging in many experimental models. To gain insights into factors affecting this reexpression of beta-MHC within the complex anatomical structure of the heart, we investigated the spatial pattern of its expression at the level of single cells during aging and hypertrophy. We generated mice that express yellow fluorescent protein fused to the N terminus of the beta-MHC and examined its expression pattern during normal aging and in mice with hypertrophy induced by constitutive expression of a renin transgene. The localization of fibrosis within the hearts also was determined by using a fluorescent lectin. The results show that reexpression of beta-MHC occurs in discrete subsets of myocytes within the subendocardium rather than uniformly throughout the heart, that beta-MHC induction is not an obligatory consequence of cellular hypertrophy, and that beta-MHC-expressing cells in the normal aging heart and the hypertrophic heart are distributed predominantly in clusters within and surrounding foci of fibrosis. We conclude that beta-MHC gene expression in the normal aging adult and hypertrophic mouse heart is a marker of fibrosis rather than of cellular hypertrophy.

To examine the mechanisms whereby amino acids modulate nitric oxide (NO) production and blood flow in the renal vasculature, chemiluminescence techniques were used to quantify NO in the renal venous effluent of the isolated, perfused rat kidney as different amino acids were added to the perfusate. The addition of 10(-4) or 10(-3) M cationic amino acids (l-ornithine, l-lysine, or l-homoarginine) or neutral amino acids (l-glutamine, l-leucine, or l-serine) to the perfusate decreased NO and increased renal vascular resistance. Perfusion with anionic amino acids (l-glutamate or l-aspartate) had no effect on either parameter. The effects of the cationic and neutral amino acids were reversed with 10(-3) M l-arginine and prevented by deendothelialization or NO synthase inhibition. The effects of the neutral amino acids but not the cationic amino acids were dependent on extracellular sodium. Cationic and neutral amino acids also decreased calcimycin-induced NO, as assessed by DAF-FM-T fluorescence, in cultured EA.hy926 endothelial cells. Inhibition of system y(+) or y(+)L by siRNA for the cationic amino acid transporter 1 or the CD98/4F2 heavy chain diminished the NO-depleting effects of these amino acids. Finally, transport studies in cultured cells demonstrated that cationic or neutral amino acids in the extracellular space stimulate efflux of l-arginine out of the cell. Thus the present experiments demonstrate that cationic and neutral amino acids can modulate NO production in endothelial cells by altering cellular l-arginine transport through y(+) and y(+)L transport mechanisms.

Lipocalin-type prostaglandin (PG) D synthase (L-PGDS) is a dually functional protein, acting both as a PGD2-synthesizing enzyme and as an extracellular transporter of various lipophilic small molecules. L-PGDS is expressed in oligodendrocytes (OLs) in the central nervous system and is up-regulated in OLs of the twitcher mouse, a model of globoid cell leukodystrophy (Krabbe's disease). We investigated whether up-regulation of L-PGDS is either unique to Krabbe's disease or is a more generalized phenomenon in lysosomal storage disorders (LSDs), using LSD mouse models of Tay-Sachs disease, Sandhoff disease, GM1 gangliosidosis and Niemann-Pick type C1 disease. Quantitative RT-PCR revealed that L-PGDS mRNA was up-regulated in the brains of all these mouse models. In addition, strong L-PGDS immunoreactivity was observed in OLs, but not in either astrocytes or microglia in these models. Thus, up-regulation of L-PGDS appears to be a common response of OLs in LSDs. Moreover, surface plasmon resonance analyses revealed that L-PGDS binds GM1 and GM2 gangliosides, accumulated in neurons in the course of LSD, with high affinities (KD = 65 and 210 nm, respectively). This suggests that L-PGDS may play a role in scavenging harmful lipophilic substrates in LSD.

To explore the effects of decreased amounts or absence of aldosterone, we have disrupted the gene coding for aldosterone synthase (AS) in mice and investigated blood pressure and kidney function in AS+/+, AS+/-, and AS-/- mice. AS+/- mice have normal blood pressures and show no abnormalities in electrolytes or kidney gene expression, but they have significantly higher than normal urine volume and lower urine osmolality. In contrast, the AS-/- mice have low blood pressure, abnormal electrolyte homeostasis (increased plasma concentrations of K+, Ca2+, and Mg2+ and decreased concentrations of HCO3(-) and Cl- but no difference in the plasma Na+ level), and disturbances in water metabolism (higher urine output, decreased urine osmolality, and impaired urine concentrating and diluting ability). Absence of aldosterone in the AS-/- mice induced several compensatory changes: an increased food intake-to-body weight ratio, an elevated plasma concentration of glucocorticoids, and strong activation of the renin-angiotensin system. Parallel with the markedly increased synthesis and release of renin, the AS-/- mice showed increased expression of cyclooxygenase-2 (COX-2) in macula densa. On salt supplementation, plasma electrolyte concentrations and kidney renin and COX-2 levels became similar to those of wild-type mice, but the lower blood pressure of the AS-/- mice was not corrected. Thus absence of aldosterone in AS-/- mice results in impairment of Na+ reabsorption in the distal nephron, decreased blood pressure, and strong renin-angiotensin system activation. Our data show the substantial correction of these abnormalities, except the low blood pressure, by high dietary salt does not depend on aldosterone.

To study the effects of decreased amounts or absence of aldosterone on development and endocrine function, we have disrupted the mouse gene, Cyp11b2, coding for aldosterone synthase (AS) by replacing its first two exons with sequences coding for enhanced green fluorescent protein. The null pups fail to thrive postnatally, and about 30% die between d 7 and 28. Aldosterone in plasma and AS mRNA in adrenal glands are undetectable in the null mice. Adult AS-null mice are small, weigh 75% of wild type, are hypotensive, have increased concentrations of plasma K(+) and corticosterone, and a decreased concentration of plasma Cl(-). Their plasma renin and angiotensin II concentrations are 45x and 4x wild type. The adrenal cortex is disorganized and has cells that contain marked accumulations of lipid. The zona glomerulosa is widened and includes easily detectable renin-containing cells, not seen in the wild-type adrenal gland. In the AS-/- adrenals, the level of mRNA for Cyp11b1, coding for 11beta-hydroxylase, is 150% wild type. The adrenal glands of the null mice consequently show evidence of a greatly activated renin-angiotensin system and up-regulation of glucocorticoid production. In the AS-null mice enhanced green fluorescent protein fluorescence is mainly at the boundary between the cortex and medulla, where apoptotic cells are numerous. These data are consistent with the absence of aldosterone in the AS-null mice inducing an increased cell-turnover of cells in the adrenals that normally become AS expressing and their migration to the medullary boundary where they apoptose.

Mice lacking Ren1c were generated using C57BL/6-derived embryonic stem cells. Mice homozygous for Ren1c disruption (Ren1c-/-) are born at the expected ratio, but approximately 80% die of dehydration within a few days. The surviving Ren1c-/- mice have no renin mRNA expression in the kidney, hydronephrosis, thickening of renal arterial walls, and fibrosis in the kidney. Plasma renin and angiotensins I and II are undetectable. Urinary aldosterone is 6% wild-type. They have low tail-cuff BP (84 +/- 4 versus 116 +/- 5 mmHg in +/+) and excrete large amounts of urine (5.2 +/- 0.8 ml/d, 725 +/- 34 mOsm versus 1.1 +/- 0.1 ml/d, 2460 +/- 170 mOsm in +/+). After 5 d of drinking 5% dextrose, desmopressin does not increase the osmolality of the urine in -/- mice (624 +/- 19 to 656 +/- 25 mOsm), whereas in +/+, it increases severalfold (583 +/- 44 to 2630 +/- 174 mOsm). Minipump infusion of angiotensin II to Ren1c-/- mice restores BP to wild-type level, but preexisting damage to the medulla prevents complete restoration of the ability of the kidney to concentrate urine. Heterozygous Ren1c+/- mice, in contrast, are indistinguishable from +/+ in BP, urine volume, and osmolality. Kidney renin mRNA, the number of kidney cells producing renin, and plasma renin concentration in the Ren1c+/- mice are also indistinguishable from +/+. These results demonstrate that renin is the only enzyme capable of maintaining plasma angiotensins and that renin expression in the kidney is very tightly regulated at the mRNA level.

Experiments were performed to determine whether L-arginine transport regulates nitric oxide (NO) production and hemodynamics in the renal medulla. The effects of renal medullary interstitial infusion of cationic amino acids, which compete with L-arginine for cellular uptake, on NO levels and blood flow in the medulla were examined in anesthetized rats. NO concentration in the renal inner medulla, measured with a microdialysis-oxyhemoglobin trapping technique, was significantly decreased by 26-44% and renal medullary blood flow, measured by laser Doppler flowmetry, was significantly reduced by 20-24% during the acute renal medullary interstitial infusion of L-ornithine, L-lysine, and L-homoarginine (1 micromol.kg(-1).min(-1) each; n = 6-8/group). In contrast, intramedullary infusion of L-arginine increased NO concentration and medullary blood flow. Flow cytometry experiments with 4-amino-5-methylamino-2',7'-difluorescein diacetate, a fluorophore reactive to intracellular NO, demonstrated that L-ornithine, L-lysine, and L-homoarginine decreased NO by 54-57% of control, whereas L-arginine increased NO by 21% in freshly isolated inner medullary cells (1 mmol/l each, n > 1,000 cells/experiment). The mRNA for the cationic amino acid transporter-1 was predominantly expressed in the inner medulla, and cationic amino acid transporter-1 protein was localized by immunohistochemistry to the collecting ducts and vasa recta in the inner medulla. These results suggest that L-arginine transport by cationic amino acid transport mechanisms is important in the production of NO and maintenance of blood flow in the renal medulla.