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Browse Health
Internist, Nephrologist (kidney)
33 years of experience
Accepting new patients

Credentials

Education ?

Medical School Score Rankings
University of Kansas (1979)
  •  
Top 50%

Awards & Distinctions ?

Associations
American Board of Internal Medicine

Affiliations ?

Dr. Brosius is affiliated with 3 hospitals.

Hospital Affiliations

Score

Rankings

  • University of Michigan Hospitals & Health Centers
    1500 E Medical Center Dr, Ann Arbor, MI 48109
    •  
    Top 25%
  • Ann Arbor Veterans Affairs Medical Center
    2215 Fuller Rd, Ann Arbor, MI 48105
  • University of Michigan Health System
  • Publications & Research

    Dr. Brosius has contributed to 96 publications.
    Title Glut1 Enhances Mtor Activity Independently of Tsc2 and Ampk.
    Date December 2011
    Journal American Journal of Physiology. Renal Physiology
    Excerpt

    Enhanced GLUT1 expression in mesangial cells plays an important role in the development of diabetic nephropathy by stimulating signaling through several pathways resulting in increased glomerular matrix accumulation. Similarly, enhanced mammalian target of rapamycin (mTOR) activation has been implicated in mesangial matrix expansion and glomerular hypertrophy in diabetes. We sought to examine whether enhanced GLUT1 expression increased mTOR activity and, if so, to identify the mechanism. We found that levels of GLUT1 expression and mTOR activation, as evidenced by S6 kinase (S6K) and 4E-BP-1 phosphorylation, changed in tandem in cell lines exposed to elevated levels of extracellular glucose. We then showed that increased GLUT1 expression enhanced S6K phosphorylation by 1.7- to 2.9-fold in cultured mesangial cells and in glomeruli from GLUT1 transgenic mice. Treatment with the mTOR inhibitor, rapamycin, eliminated the GLUT1 effect on S6K phosphorylation. In cells lacking functional tuberous sclerosis complex (TSC) 2, GLUT1 effects on mTOR activity persisted, indicating that GLUT1 effects were not mediated by TSC. Similarly, AMP kinase activity was not altered by enhanced GLUT1 expression. Conversely, enhanced GLUT1 expression led to a 2.4-fold increase in binding of mTOR to its activator, Rheb, and a commensurate 2.1-fold decrease in binding of Rheb to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) consistent with mediation of GLUT1 effects by a metabolic effect on GAPDH. Thus, GLUT1 expression appears to augment mesangial cell growth and matrix protein accumulation via effects on glycolysis and decreased GAPDH interaction with Rheb.

    Title Understanding Kidney Disease: Toward the Integration of Regulatory Networks Across Species.
    Date February 2011
    Journal Seminars in Nephrology
    Excerpt

    Animal models have long been useful in investigating both normal and abnormal human physiology. Systems biology provides a relatively new set of approaches to identify similarities and differences between animal models and human beings that may lead to a more comprehensive understanding of human kidney pathophysiology. In this review, we briefly describe how genome-wide analyses of mouse models have helped elucidate features of human kidney diseases, discuss strategies to achieve effective network integration, and summarize currently available web-based tools that may facilitate integration of data across species. The rapid progress in systems biology and orthology, as well as the advent of web-based tools to facilitate these processes, now make it possible to take advantage of knowledge from distant animal species in targeted identification of regulatory networks that may have clinical relevance for human kidney diseases.

    Title Susceptible Mice: Identifying a Diabetic Nephropathy Disease Locus Using a Murine Model.
    Date November 2010
    Journal Kidney International
    Excerpt

    Diabetic nephropathy is a common, complex disease with a clear genetic predisposition. Human gene association studies are beginning to bear fruit by identifying gene loci that increase diabetic nephropathy risk. Chua et al. report a similar study in diabetic mice that reveals a major nephropathy locus on chromosome 8. Could this be a human nephropathy gene? Time will tell, but such findings will at least improve the use of mouse models of human kidney disease.

    Title Sex Dimorphic Actions of Rosiglitazone in Generalised Peroxisome Proliferator-activated Receptor-gamma (ppar-gamma)-deficient Mice.
    Date August 2010
    Journal Diabetologia
    Excerpt

    The aim of this study was to determine the dependency on peroxisome proliferator-activated receptor-gamma (PPAR-gamma) of insulin sensitisation and glucose homeostasis by thiazolidinediones using a global Ppar-gamma (also known as Pparg)-knockout mouse model.

    Title Podocyte-specific Overexpression of Glut1 Surprisingly Reduces Mesangial Matrix Expansion in Diabetic Nephropathy in Mice.
    Date July 2010
    Journal American Journal of Physiology. Renal Physiology
    Excerpt

    Increased expression of the facilitative glucose transporter, GLUT1, leads to glomerulopathy that resembles diabetic nephropathy, whereas prevention of enhanced GLUT1 expression retards nephropathy. While many of the GLUT1-mediated effects are likely due to mesangial cell effects, we hypothesized that increased GLUT1 expression in podocytes also contributes to the progression of diabetic nephropathy. Therefore, we generated two podocyte-specific GLUT1 transgenic mouse lines (driven by a podocin promoter) on a db/m C57BLKS background. Progeny of the two founders were used to generate diabetic db/db and control db/m littermate mice. Immunoblots of glomerular lysates showed that transgenic mice had a 3.5-fold (line 1) and 2.1-fold (line 2) increase in GLUT1 content compared with wild-type mice. Both lines showed similar increases in fasting blood glucose and body weights at 24 wk of age compared with wild-type mice. Mesangial index (percent PAS-positive material in the mesangial tuft) increased 88% (line 1) and 75% (line 2) in the wild-type diabetic mice but only 48% (line 1) and 39% (line 2) in the diabetic transgenic mice (P < 0.05, transgenic vs. wild-type mice). This reduction in mesangial expansion was accompanied by a reduction in fibronectin accumulation, and vascular endothelial growth factor (VEGF) levels increased only half as much in the transgenic diabetic mice as in wild-type diabetic mice. Levels of nephrin, neph1, CD2AP, podocin, and GLUT4 were not significantly different in transgenic compared with wild-type mice. Taken together, increased podocyte GLUT1 expression in diabetic mice does not contribute to early diabetic nephropathy; surprisingly, it protects against mesangial expansion and fibronectin accumulation possibly by blunting podocyte VEGF increases.

    Title A New Pair of Socs for Diabetic Nephropathy.
    Date June 2010
    Journal Journal of the American Society of Nephrology : Jasn
    Title The Management of Diabetic Neuropathy in Ckd.
    Date February 2010
    Journal American Journal of Kidney Diseases : the Official Journal of the National Kidney Foundation
    Title Mouse Models of Diabetic Nephropathy.
    Date December 2009
    Journal Journal of the American Society of Nephrology : Jasn
    Excerpt

    Diabetic nephropathy is a major cause of ESRD worldwide. Despite its prevalence, a lack of reliable animal models that mimic human disease has delayed the identification of specific factors that cause or predict diabetic nephropathy. The Animal Models of Diabetic Complications Consortium (AMDCC) was created in 2001 by the National Institutes of Health to develop and characterize models of diabetic nephropathy and other complications. This interim report and our online supplement detail the progress made toward that goal, specifically in the development and testing of murine models. Updates are provided on validation criteria for early and advanced diabetic nephropathy, phenotyping methods, the effect of background strain on nephropathy, current best models of diabetic nephropathy, negative models, and views of future directions. AMDCC investigators and other investigators in the field have yet to validate a complete murine model of human diabetic kidney disease. Nonetheless, the critical analysis of existing murine models substantially enhances our understanding of this disease process.

    Title Glut1-induced Cflip Expression Promotes Proliferation and Prevents Apoptosis in Vascular Smooth Muscle Cells.
    Date October 2009
    Journal American Journal of Physiology. Cell Physiology
    Excerpt

    Enhanced expression of the facilitative glucose transporter, GLUT1, has been shown to inhibit apoptosis in several cell systems including vascular smooth muscle cells (VSMCs). A decrease in apoptosis could lead to increased VSMC numbers in neointimal and medial arterial layers under several pathologic conditions. The hypothesis underlying these studies is that GLUT1 induces expression of antiapoptotic and prosurvival genes that increase VSMC survival. Transcriptomic analysis of A7r5 VSMCs, in which GLUT1 was acutely overexpressed, showed a 2.14-fold increase in c-FLICE inhibitory protein (cFLIP), which promotes cellular growth and prevents apoptosis through caspase 8 binding. We confirmed that overexpression of GLUT1 induced mRNA and protein expression of both the long and short isoforms of cFLIP (cFLIP(L) and cFLIP(S)) in primary and stable immortalized VSMC lines as well as in aortas from GLUT1 transgenic mice. Increased GLUT1 reduced VSMC death by more than twofold after serum withdrawal, as evidenced by decreased caspase 3 activity and Trypan blue exclusion studies. GLUT1 overexpression resulted in a greater than twofold increase in proliferating cell nuclear antigen expression and live cell numbers, consistent with augmented VSMC proliferation. Lentiviral knockdown of cFLIP(L) showed that cFLIP(L) was necessary for the proproliferative and antiapoptotic effects of GLUT1 overexpression. Taken together, these data suggest that GLUT1 induction of cFLIP(L) expression augments proliferation and prevents apoptosis in VSMCs.

    Title The Peroxisome Proliferator-activated Receptor Gamma Agonist Pioglitazone Improves Cardiometabolic Risk and Renal Inflammation in Murine Lupus.
    Date September 2009
    Journal Journal of Immunology (baltimore, Md. : 1950)
    Excerpt

    Individuals with systemic lupus erythematosus (SLE) have a striking increase in the risk of premature atherosclerosis, a complication preceded by significant subclinical vascular damage. A proposed mechanism leading to accelerated vascular disease in SLE is an imbalance between vascular damage and repair, as patients with this disease display significant abnormalities in phenotype and function of endothelial progenitor cells. In addition, individuals with SLE have a higher incidence of insulin resistance which may further contribute to the increased cardiovascular risk. This study examined the role of the peroxisome proliferator activated receptor gamma agonist pioglitazone in improving endothelial function, endothelial progenitor cell numbers and functional capacity, metabolic parameters, and disease activity in the lupus-prone murine model New Zealand Black/New Zealand White (NZB x NZW)F(1). Ten-week-old prenephritic female NZB/NZW F(1) mice were exposed to 10 or 25 mg/kg/day of oral pioglitazone or vehicle for 15 or 24 wk. Mice exposed to pioglitazone exhibited pronounced enhancement in endothelial-dependent vasorelaxation of thoracic aortas and in endothelial progenitor cell function, as assessed by the capacity of bone marrow-derived endothelial progenitor cells to differentiate into mature endothelial cells. Pioglitazone-treated mice showed improvement in insulin resistance, adipokine, and lipid profile. Kidneys from pioglitazone-treated mice showed significant decreases in immune complex deposition, renal inflammation, T cell glomerular infiltration, and intrarenal synthesis of TNF-alpha, IL-1beta, and VCAM-1. These results indicate that peroxisome proliferator-activated receptor gamma agonists could serve as important tools in the prevention of premature cardiovascular disease and organ damage in SLE.

    Title A Rapid, Ppar-gamma-dependent Effect of Pioglitazone on the Phosphorylation of Mypt.
    Date June 2009
    Journal American Journal of Physiology. Cell Physiology
    Excerpt

    Peroxisome proliferator-activated receptor (PPAR)-gamma ligands, thiazolidinediones, have been demonstrated to regulate vascular reactivity. We examined the effect of pioglitazone (PIO; 20 muM) in rat primary cultured aortic smooth muscle cells on constitutive phosphorylation of the regulatory subunit of myosin phosphatase (MYPT). PIO decreased the phosphorylation of Thr(697) on MYPT within 15 min, and the inhibition was maintained up to 6 h. The PPAR-gamma antagonist GW-9662 (5 microM) abrogated the inhibition of Thr(697) phosphorylation mediated by PIO. Because longer-term PIO treatment inhibits RhoA/Rho kinase (ROCK) signaling and Thr(697) phosphorylation, we tested the effect of the ROCK inhibitor Y-27632 (10 muM) on the inhibition of Thr(697) phosphorylation by PIO. Y-27632 alone inhibited Thr(697) phosphorylation, and there was an additive effect with PIO. In addition, up to 1 h of PIO treatment did not affect RhoA localization or decrease ROCK-dependent phosphorylation of Thr(855). These results suggest that the effect of PIO is independent of inhibition of RhoA/ROCK. PIO increased the phosphorylation of Ser(696) in the same time course as its effect on Thr(697). Ser(696) has been shown to be phosphorylated by PKA and PKG. PKA inhibitor H-89 (10 microM) and PKG inhibitor KT-5823 (0.5 microM) abrogated the effect of PIO on both Thr(697) and Ser(696) phosphorylation. The constitutive turnover of phosphorylation of Thr(697) is rapid, suggesting that the decreased phosphorylation of Thr(697) by PIO is due to enhanced phosphorylation of Ser(696). This is supported by the finding that PIO blocks ANG II-stimulated phosphorylation of Thr(697) but not ANG II-stimulated RhoA translocation. Therefore, the effect of shorter-term PIO apparently is to increase myosin light chain phosphatase activity, thereby desensitizing the vascular smooth muscle to agonist signaling.

    Title Enhanced Expression of Janus Kinase-signal Transducer and Activator of Transcription Pathway Members in Human Diabetic Nephropathy.
    Date March 2009
    Journal Diabetes
    Excerpt

    Glomerular mesangial expansion and podocyte loss are important early features of diabetic nephropathy, whereas tubulointerstitial injury and fibrosis are critical for progression of diabetic nephropathy to kidney failure. Therefore, we analyzed the expression of genes in glomeruli and tubulointerstitium in kidney biopsies from diabetic nephropathy patients to identify pathways that may be activated in humans but not in murine models of diabetic nephropathy that fail to progress to glomerulosclerosis, tubulointerstitial fibrosis, and kidney failure.

    Title New Insights into the Mechanisms of Fibrosis and Sclerosis in Diabetic Nephropathy.
    Date January 2009
    Journal Reviews in Endocrine & Metabolic Disorders
    Excerpt

    Progression of diabetic nephropathy (DN) is manifested by gradual scarring of both the renal glomerulus and tubulointerstitial region. Over the past several years, the general understanding of the pathogenic factors that lead to renal fibrosis in DN has expanded considerably. In this review, some of the important factors that appear to be involved in driving this fibrosing process are discussed, with special emphasis on newer findings and insights. It is now clear that multiple cell types in the kidney contribute to progressive fibrosis in DN. New concepts about bradykinin, TGF-beta and eNOS signaling as well as JAK/STAT activation and the central role of inflammation in both glomerular and tubulointerstitial fibrosis are discussed.

    Title Rosiglitazone Reduces Renal and Plasma Markers of Oxidative Injury and Reverses Urinary Metabolite Abnormalities in the Amelioration of Diabetic Nephropathy.
    Date December 2008
    Journal American Journal of Physiology. Renal Physiology
    Excerpt

    Recent studies suggest that thiazolidinediones ameliorate diabetic nephropathy (DN) independently of their effect on hyperglycemia. In the current study, we confirm and extend these findings by showing that rosiglitazone treatment prevented the development of DN and reversed multiple markers of oxidative injury in DBA/2J mice made diabetic by low-dose streptozotocin. These diabetic mice developed a 14.2-fold increase in albuminuria and a 53% expansion of renal glomerular extracellular matrix after 12 wk of diabetes. These changes were largely abrogated by administration of rosiglitazone beginning 2 wk after the completion of streptozotocin injections. Rosiglitazone had no effect on glycemic control. Rosiglitazone had similar effects on insulin-treated diabetic mice after 24 wk of diabetes. Podocyte loss and glomerular fibronectin accumulation, other markers of early DN, were prevented by rosiglitazone in both 12- and 24-wk diabetic models. Surprisingly, glomerular GLUT1 levels did not increase and nephrin levels did not decrease in the diabetic animals; neither changed with rosiglitazone. Plasma and kidney markers of protein oxidation and lipid peroxidation were significantly elevated in the 24-wk diabetic animals despite insulin treatment and were reduced to near-normal levels by rosiglitazone. Finally, urinary metabolites were markedly altered by diabetes. Of 1,988 metabolite features identified by electrospray ionization time of flight mass spectrometry, levels of 56 were altered more than twofold in the urine of diabetic mice. Of these, 21 were returned to normal by rosiglitazone. Thus rosiglitazone has direct effects on the renal glomerulus to reduce reactive oxygen species accumulation to prevent type 1 diabetic mice from development of DN.

    Title Is the Er Stressed out in Diabetic Kidney Disease?
    Date November 2008
    Journal Journal of the American Society of Nephrology : Jasn
    Title Rosiglitazone Treatment Reduces Diabetic Neuropathy in Streptozotocin-treated Dba/2j Mice.
    Date November 2008
    Journal Endocrinology
    Excerpt

    Diabetic neuropathy (DN) is a common complication of diabetes. Currently, there is no drug treatment to prevent or slow the development of DN. Rosiglitazone (Rosi) is a potent insulin sensitizer and may also slow the development of DN by a mechanism independent of its effect on hyperglycemia. A two by two design was used to test the effect of Rosi treatment on the development of DN. Streptozotocin-induced diabetic DBA/2J mice were treated with Rosi. DN and oxidative stress were quantified, and gene expression was profiled using the Affymetrix Mouse Genome 430 2.0 microarray platform. An informatics approach identified key regulatory elements activated by Rosi. Diabetic DBA/2J mice developed severe hyperglycemia, DN, and elevated oxidative stress. Rosi treatment did not affect hyperglycemia but did reduce oxidative stress and prevented the development of thermal hypoalgesia. Two novel transcription factor binding modules were identified that may control genes correlated to changes in DN after Rosi treatment: SP1F_ZBPF and EGRF_EGRF. These targets may be useful in designing drugs with the same efficacy as Rosi in treating DN but with fewer undesirable effects.

    Title A Gsk-3/tsc2/mtor Pathway Regulates Glucose Uptake and Glut1 Glucose Transporter Expression.
    Date October 2008
    Journal American Journal of Physiology. Cell Physiology
    Excerpt

    Glucose transport is a highly regulated process and is dependent on a variety of signaling events. Glycogen synthase kinase-3 (GSK-3) has been implicated in various aspects of the regulation of glucose transport, but the mechanisms by which GSK-3 activity affects glucose uptake have not been well defined. We report that basal glycogen synthase kinase-3 (GSK-3) activity regulates glucose transport in several cell types. Chronic inhibition of basal GSK-3 activity (8-24 h) in several cell types, including vascular smooth muscle cells, resulted in an approximately twofold increase in glucose uptake due to a similar increase in protein expression of the facilitative glucose transporter 1 (GLUT1). Conversely, expression of a constitutively active form of GSK-3beta resulted in at least a twofold decrease in GLUT1 expression and glucose uptake. Since GSK-3 can inhibit mammalian target of rapamycin (mTOR) signaling via phosphorylation of the tuberous sclerosis complex subunit 2 (TSC2) tumor suppressor, we investigated whether chronic GSK-3 effects on glucose uptake and GLUT1 expression depended on TSC2 phosphorylation and TSC inhibition of mTOR. We found that absence of functional TSC2 resulted in a 1.5-to 3-fold increase in glucose uptake and GLUT1 expression in multiple cell types. These increases in glucose uptake and GLUT1 levels were prevented by inhibition of mTOR with rapamycin. GSK-3 inhibition had no effect on glucose uptake or GLUT1 expression in TSC2 mutant cells, indicating that GSK-3 effects on GLUT1 and glucose uptake were mediated by a TSC2/mTOR-dependent pathway. The effect of GSK-3 inhibition on GLUT1 expression and glucose uptake was restored in TSC2 mutant cells by transfection of a wild-type TSC2 vector, but not by a TSC2 construct with mutated GSK-3 phosphorylation sites. Thus, TSC2 and rapamycin-sensitive mTOR function downstream of GSK-3 to modulate effects of GSK-3 on glucose uptake and GLUT1 expression. GSK-3 therefore suppresses glucose uptake via TSC2 and mTOR and may serve to match energy substrate utilization to cellular growth.

    Title Podocyte Specific Knock out of Selenoproteins Does Not Enhance Nephropathy in Streptozotocin Diabetic C57bl/6 Mice.
    Date September 2008
    Journal Bmc Nephrology
    Excerpt

    BACKGROUND: Selenoproteins contain selenocysteine (Sec), commonly considered the 21st genetically encoded amino acid. Many selenoproteins, such as the glutathione peroxidases and thioredoxin reductases, protect cells against oxidative stress by functioning as antioxidants and/or through their roles in the maintenance of intracellular redox balance. Since oxidative stress has been implicated in the pathogenesis of diabetic nephropathy, we hypothesized that selenoproteins protect against this complication of diabetes. METHODS: C57BL/6 mice that have a podocyte-specific inability to incorporate Sec into proteins (denoted in this paper as PodoTrsp-/-) and control mice were made diabetic by intraperitoneal injection of streptozotocin, or were injected with vehicle. Blood glucose, body weight, microalbuminuria, glomerular mesangial matrix expansion, and immunohistochemical markers of oxidative stress were assessed. RESULTS: After 3 and 6 months of diabetes, control and PodoTrsp-/- mice had similar levels of blood glucose. There were no differences in urinary albumin/creatinine ratios. Periodic acid-Schiff staining to examine mesangial matrix expansion also demonstrated no difference between control and PodoTrsp-/- mice after 6 months of diabetes, and there were no differences in immunohistochemical stainings for nitrotyrosine or NAD(P)H dehydrogenase, quinone 1. CONCLUSION: Loss of podocyte selenoproteins in streptozotocin diabetic C57BL/6 mice does not lead to increased oxidative stress as assessed by nitrotyrosine and NAD(P)H dehydrogenase, quinone 1 immunostaining, nor does it lead to worsening nephropathy.

    Title From Fibrosis to Sclerosis: Mechanisms of Glomerulosclerosis in Diabetic Nephropathy.
    Date July 2008
    Journal Diabetes
    Title Hypoxia-inducible Factor-1alpha is a Critical Mediator of Hypoxia Induced Apoptosis in Cardiac H9c2 and Kidney Epithelial Hk-2 Cells.
    Date June 2008
    Journal Bmc Cardiovascular Disorders
    Excerpt

    BACKGROUND: Hypoxia inducible factor-1 (HIF-1) is a transcription factor that functions to maintain cellular homeostasis in response to hypoxia. There is evidence that HIF-1 can also trigger apoptosis, possibly when cellular responses are inadequate to meet energy demands under hypoxic conditions. METHODS: Cardiac derived H9c2 and renal tubular epithelial HK-2 cells expressing either the wild type oxygen regulated subunit of HIF-1 (pcDNA3-Hif-1alpha) or a dominant negative version that lacked both DNA binding and transactivation domains (pcDNA3-DN-Hif-1alpha), were maintained in culture and exposed to hypoxia. An RNA interference approach was also employed to selectively knockdown expression of Hif-1alpha. Apoptosis was analyzed in both H9c2 and HK-2 cells by Hoechst and TUNEL staining, caspase 3 activity assays and activation of pro-apoptotic Bcl2 family member Bax. RESULTS: Overexpression of pcDNA3-DN-Hif-1alpha led to a significant reduction in hypoxia -induced apoptosis (17 +/- 2%, P < 0.01) in H9c2 cells compared to both control-transfected and wild type Hif-1alpha transfected cells. Moreover, selective ablation of HIF-1alpha protein expression by RNA interference in H9c2 cells led to 55% reduction of caspase 3 activity and 46% reduction in the number of apoptotic cells as determined by Hoechst 33258 staining, after hypoxia. Finally, upregulation of the pro-apoptotic protein, Bax, was found in H9c2 cells overexpressing full-length pcDNA3-HA-HIF-1alpha exposed to hypoxia. In HK-2 cells overexpression of wild-type Hif-1alpha led to a two-fold increase in Hif-1alpha levels during hypoxia. This resulted in a 3.4-fold increase in apoptotic cells and a concomitant increase in caspase 3 activity during hypoxia when compared to vector transfected control cells. HIF-1alpha also induced upregulation of Bax in HK-2 cells. In addition, introduction of dominant negative Hif-1alpha constructs in both H9c2 and HK-2 -cells led to decreased active Bax expression. CONCLUSION: These data demonstrate that HIF-1alpha is an important component of the apoptotic signaling machinery in the two cell types.

    Title Role for Glut1 in Diabetic Glomerulosclerosis.
    Date October 2007
    Journal Expert Reviews in Molecular Medicine
    Excerpt

    Numerous studies have investigated specific pathways that link diabetes and high extracellular glucose exposure to glomerulosclerosis and mesangial cell extracellular matrix production. However, only in the past ten years has a role for glucose transporters in this process been addressed. Many different glucose transporters are expressed in glomeruli; of these, the GLUT1 facilitative glucose transporter is upregulated in the diabetic renal cortex and in response to glomerular hypertension, as well as in cultured mesangial cells exposed to high glucose. Transgenic mouse and cell models have recently been developed to test the role of GLUT1 in the pathogenesis of glomerulosclerosis with and without diabetes. Clinical studies of GLUT1 alleles performed in humans have identified GLUT1 susceptibility alleles for diabetic nephropathy. Studies are also currently under way to assess the potential role of GLUT1 in nondiabetic renal disorders.

    Title Preserved Expression of Glut4 Prevents Enhanced Agonist-induced Vascular Reactivity and Mypt1 Phosphorylation in Hypertensive Mouse Aorta.
    Date September 2007
    Journal American Journal of Physiology. Heart and Circulatory Physiology
    Excerpt

    We previously showed that GLUT4 expression is decreased in arterial smooth muscle of deoxycorticosterone acetate (DOCA)-salt hypertensive rats and that GLUT4-knockout mice have enhanced arterial reactivity. Therefore, we hypothesized that increased GLUT4 expression in vascular smooth muscle in vivo would prevent enhanced arterial reactivity and possibly reduce blood pressure in DOCA-salt hypertensive mice. Adult wild-type (WT) and GLUT4 transgenic (TG) mice were subjected to DOCA-salt hypertension with uninephrectomy or underwent uninephrectomy and remained normotensive. GLUT4 expression was increased more than twofold in the aortas of GLUT4 TG mice compared with WT aortas. Eight weeks after implantation of the DOCA pellets, GLUT4 expression decreased by 75% in aortas of WT hypertensive mice, but not in GLUT4 TG hypertensive aortas. Systolic blood pressure was significantly and similarly increased in WT and GLUT4 TG DOCA-salt mice compared with their respective sham-treated controls (159 vs. 111 mmHg). Responsiveness to the contractile agonist 5-HT was significantly increased in aortic rings from WT DOCA-salt mice but remained normal in GLUT4 TG DOCA mice. Phosphorylation of the myosin phosphatase targeting subunit MYPT1 was significantly enhanced in aortas of WT DOCA-salt mice, and this increase was prevented in GLUT4 TG mice. MYPT1 phosphorylation was also increased in nonhypertensive GLUT4-knockout mice. Myosin phosphatase, a major negative regulator of calcium sensitivity, is itself negatively regulated by phosphorylation of MYPT1. Therefore, our results show that preservation of GLUT4 expression prevents enhanced arterial reactivity in hypertension, possibly via effects on myosin phosphatase activity.

    Title Identification of the Glomerular Podocyte As a Target for Growth Hormone Action.
    Date June 2007
    Journal Endocrinology
    Excerpt

    GH excess in both the human and transgenic animal models is characterized by significant changes in blood pressure and renal function. The GH/GH receptor (GHR) axis is also implicated in the development of diabetic nephropathy. However, it is not clear whether GH's actions on renal function are due to indirect actions mediated via changes in blood pressure and vascular tone or due to direct action of GH on the kidney. We hypothesized that functional GHRs are expressed on the glomerular podocyte enabling direct actions of GH on glomerular function. Real-time PCR, immunohistochemistry, and Western blot analysis of murine podocyte cells (MPC-5) and kidney glomeruli demonstrated expression of GHR mRNA and protein. Exposure of both murine and human podocytes to GH (50-500 ng/ml) resulted in an increase in abundance of phosphorylated signal transducer and activator of transcription-5, Janus kinase-2, and ERK1/2 proteins. Exposure of podocytes to GH also caused changes in the intracellular distribution of the Janus kinase-2 adapter protein Src homology 2-Bbeta, stimulation of focal adhesion kinase, increase in reactive oxygen species, and GH-dependent changes in the actin cytoskeleton. We conclude that glomerular podocytes express functional GHRs and that GH increases levels of reactive oxygen species and induces reorganization of the actin cytoskeleton in these cells. These results provide a novel mechanistic link between GH's actions and glomerular dysfunction in disorders such as acromegaly and diabetic glomerulosclerosis.

    Title Hypotension, Lipodystrophy, and Insulin Resistance in Generalized Ppargamma-deficient Mice Rescued from Embryonic Lethality.
    Date May 2007
    Journal The Journal of Clinical Investigation
    Excerpt

    We rescued the embryonic lethality of global PPARgamma knockout by breeding Mox2-Cre (MORE) mice with floxed PPARgamma mice to inactivate PPARgamma in the embryo but not in trophoblasts and created a generalized PPARgamma knockout mouse model, MORE-PPARgamma knockout (MORE-PGKO) mice. PPARgamma inactivation caused severe lipodystrophy and insulin resistance; surprisingly, it also caused hypotension. Paradoxically, PPARgamma agonists had the same effect. We showed that another mouse model of lipodystrophy was hypertensive, ruling out the lipodystrophy as a cause. Further, high salt loading did not correct the hypotension in MORE-PGKO mice. In vitro studies showed that the vasculature from MORE-PGKO mice was more sensitive to endothelial-dependent relaxation caused by muscarinic stimulation, but was not associated with changes in eNOS expression or phosphorylation. In addition, vascular smooth muscle had impaired contraction in response to alpha-adrenergic agents. The renin-angiotensin-aldosterone system was mildly activated, consistent with increased vascular capacitance or decreased volume. These effects are likely mechanisms contributing to the hypotension. Our results demonstrated that PPARgamma is required to maintain normal adiposity and insulin sensitivity in adult mice. Surprisingly, genetic loss of PPARgamma function, like activation by agonists, lowered blood pressure, likely through a mechanism involving increased vascular relaxation.

    Title Detection of Chronic Kidney Disease in Patients with or at Increased Risk of Cardiovascular Disease: a Science Advisory from the American Heart Association Kidney And Cardiovascular Disease Council; the Councils on High Blood Pressure Research, Cardiovascular Disease in the Young, and Epidemiology and Prevention; and the Quality of Care and Outcomes Research Interdisciplinary Working Group: Developed in Collaboration with the National Kidney Foundation.
    Date October 2006
    Journal Circulation
    Excerpt

    Chronic kidney disease (CKD) occurs commonly in patients with cardiovascular disease. In addition, CKD is a risk factor for the development and progression of cardiovascular disease. In this advisory, we present recommendations for the detection of CKD in patients with cardiovascular disease. CKD can be reliably detected with the combined use of the Modification of Diet in Renal Disease equation to estimate glomerular filtration rate and a sensitive test to detect microalbuminuria. All patients with cardiovascular disease should be screened for evidence of kidney disease with these two determinations.

    Title Detection of Chronic Kidney Disease in Patients with or at Increased Risk of Cardiovascular Disease: a Science Advisory from the American Heart Association Kidney and Cardiovascular Disease Council; the Councils on High Blood Pressure Research, Cardiovascular Disease in the Young, and Epidemiology and Prevention; and the Quality of Care and Outcomes Research Interdisciplinary Working Group: Developed in Collaboration With the National Kidney Foundation.
    Date October 2006
    Journal Hypertension
    Excerpt

    Chronic kidney disease (CKD) occurs commonly in patients with cardiovascular disease. In addition, CKD is a risk factor for the development and progression of cardiovascular disease. In this advisory, we present recommendations for the detection of CKD in patients with cardiovascular disease. CKD can be reliably detected with the combined use of the Modification of Diet in Renal Disease equation to estimate glomerular filtration rate and a sensitive test to detect microalbuminuria. All patients with cardiovascular disease should be screened for evidence of kidney disease with these two determinations.

    Title Reduction in Podocyte Density As a Pathologic Feature in Early Diabetic Nephropathy in Rodents: Prevention by Lipoic Acid Treatment.
    Date May 2006
    Journal Bmc Nephrology
    Excerpt

    A reduction in the number of podocytes and podocyte density has been documented in the kidneys of patients with diabetes mellitus. Additional studies have shown that podocyte injury and loss occurs in both diabetic animals and humans. However, most studies in animals have examined relatively long-term changes in podocyte number and density and have not examined effects early after initiation of diabetes. We hypothesized that streptozotocin diabetes in rats and mice would result in an early reduction in podocyte density and that this reduction would be prevented by antioxidants.

    Title Glut4 Facilitative Glucose Transporter Specifically and Differentially Contributes to Agonist-induced Vascular Reactivity in Mouse Aorta.
    Date December 2005
    Journal Arteriosclerosis, Thrombosis, and Vascular Biology
    Excerpt

    OBJECTIVE: We hypothesized that GLUT4 is a predominant facilitative glucose transporter in vascular smooth muscle cells (VSMCs), and GLUT4 is necessary for agonist-induced VSMC contraction. METHODS AND RESULTS: Glucose deprivation and indinavir, a GLUT4 antagonist, were used to assess the role of GLUT4 and non-GLUT4 transporters in vascular reactivity. In isolated endothelium-denuded mouse aorta, approximately 50% of basal glucose uptake was GLUT4-dependent. Norepinephrine-mediated contractions were dependent on both GLUT4 and non-GLUT4 transporters, serotonin (5-HT)-mediated contractions were mainly GLUT4-dependent, and prostaglandin (PG) F(2alpha)-mediated contractions were dependent on non-GLUT4 transporters, whereas indinavir had no effect in GLUT4 knockout vessels. We also observed a 46% decrease in GLUT4 expression in aortas from angiotensin II hypertensive mice. Indinavir caused a less profound attenuation of maximal 5-HT-mediated contraction in these vessels, corresponding to the lower GLUT4 levels in the hypertensive aortas. Finally, and somewhat surprisingly, chronic GLUT4 knockout was associated with increased vascular reactivity compared with that in wild-type animals, suggesting that chronic absence or reduction of GLUT4 expression in VSMCs leads to opposite effects observed with acute inhibition of GLUT4. CONCLUSIONS: Thus, we conclude that GLUT4 is constitutively expressed in large arteries and likely participates in basal glucose uptake. In addition, GLUT4, as well as other non-GLUT4 facilitative glucose transporters, are necessary for agonist-induced contraction, but each transporter participates in VSMC contraction selectively, depending on the agonist, and changes in GLUT4 expression may account for some of the functional changes associated with vascular diseases like hypertension.

    Title Increased Jnk Phosphorylation and Oxidative Stress in Response to Increased Glucose Flux Through Increased Glut1 Expression in Rat Retinal Endothelial Cells.
    Date November 2005
    Journal Investigative Ophthalmology & Visual Science
    Excerpt

    PURPOSE: To investigate whether increased glucose flux through increased glucose transporter1 (GLUT1) expression results in increased oxidative stress and increased c-jun N-terminal kinase (JNK) phosphorylation. METHODS: GLUT1-overexpressing cells were established using a rat retinal endothelial cell line. The intracellular reactive oxygen species was detected by the oxidation of 5- (and -6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2-DCFDA). Western blot was performed to determine JNK phosphorylation and lipid peroxidation. Differentially expressed genes were detected by cDNA microarray analysis and confirmed by Northern blot analysis. RESULTS: Clones overexpressing GLUT1 showed an approximate four- to eightfold increase in GLUT1 expression and a 44% increase in intracellular glucose concentrations. GLUT1-overexpressing cells had a 80% increase in DCF fluorescence and increased lipid peroxidation, as well as increased JNK phosphorylation. Analysis of differentially expressed genes in GLUT1-overexpressing cells showed increased expression of JNK interacting protein (JIP)-1, a scaffold protein necessary for JNK activation. Northern blot analysis confirmed upregulation of JIP-1. Immunoprecipitation showed that phosphorylated JNK, but not total JNK, coimmunoprecipitated with JIP-1 protein. At the cellular level, JIP-1 was predominantly localized in cytoplasm, especially in the perinuclear area in retinal endothelial cells. CONCLUSIONS: GLUT1 overexpression and increased glucose flux result in increased oxidative stress and JNK phosphorylation in immortalized rat retinal endothelial cells. Further studies are needed to understand molecular events after increased glucose flux in retinal endothelial cells and the relation between increased oxidative stress and JNK phosphorylation.

    Title Diabetic Nephropathy: of Mice and Men.
    Date August 2005
    Journal Advances in Chronic Kidney Disease
    Excerpt

    Accumulating evidence supports intrinsic genetic susceptibility as an important variable in the progression of diabetic nephropathy in people. Mice provide an experimental platform of unparalleled power for dissecting the genetics of mammalian diseases; however, phenotypic analysis of diabetic mice lags behind that already established for humans. Standardized benchmarks of hyperglycemia, albuminuria, and measurements of renal failure remain to be developed for different inbred strains of mice. The most glaring deficiency has been the lack of a diabetic mouse model that develops progressively worsening renal insufficiency, the sine qua non of diabetic nephropathy in humans. Differences in susceptibility of these inbred strains to complications of diabetes mellitus provide a possible avenue to dissect the genetic basis of diabetic nephropathy; however, the identification of those strains and/or mutants most susceptible to renal injury from diabetes mellitus is lacking. Identification of a mouse model that faithfully mirrors the pathogenesis of DN in humans will undoubtedly facilitate the development of new diagnostic and therapeutic interventions.

    Title Immunolocalization of the Proton-coupled Oligopeptide Transporter Pept2 in Developing Rat Brain.
    Date July 2005
    Journal Molecular Pharmaceutics
    Excerpt

    This study examined the tissue distribution, cellular localization, and developmental expression of the PEPT2 protein in rat brain. Immunoblot and immunocytochemistry analyses were performed with specific rat PEPT1 and PEPT2 antisera developed in our laboratory. Rats were examined from fetus (gestation for 17 days) to adult (day 75). On immunoblot analysis, the PEPT2 protein was detected in cerebral cortex, olfactory bulb, basal ganglia, cerebellum, and hindbrain sections of adult brain, with the strongest signals in cerebral cortex. No PEPT1 protein was found in brain. Expression levels of the PEPT2 protein in cerebral cortex were maximal in the fetus and declined rapidly with advancing age. Adult protein levels were approximately 14% of that observed in fetus. In immunofluorescence experiments, the strongest PEPT2 signals were observed in epithelial cells of the choroid plexus for both adult and neonate brains. The PEPT2 protein was exclusively expressed on the apical membrane (CSF-facing) of choroid plexus epithelia. In double labeling experiments, PEPT2 immunoreactivity in adult brain colocalized with NeuN, a neuronal marker, but not with GFAP, an astrocyte marker. In contrast, in neonatal brain, PEPT2 immunoreactivity colocalized with both GFAP and NeuN. These findings demonstrate that the PEPT2 protein is found throughout the brain. The apical expression of PEPT2 in choroid plexus suggests that it is involved in the export of neuropeptides, peptide fragments, and peptide-like drugs from cerebrospinal fluid. PEPT2 may also play a role in the regulation of neuropeptide concentrations in extracellular fluid, especially during early development.

    Title Glucose Transporters in Diabetic Nephropathy.
    Date June 2005
    Journal Pediatric Nephrology (berlin, Germany)
    Excerpt

    Changes in glucose transporter expression in glomerular cells occur early in diabetes. These changes, especially the GLUT1 increase in mesangial cells, appear to play a pathogenic role in the development of ECM expansion and perhaps other features of diabetic nephropathy. In addition, it appears that at least some diabetic patients may be predisposed to nephropathy because of polymorphisms in their GLUT1 genes. GLUT1 overexpression leads to increased glucose metabolic flux which in turn triggers the polyol pathway and activation of PKC alpha and B1. Activation of these PKC isoforms can lead directly to AP-1 induced increases in fibronectin expression and ECM accumulation. Other, more novel effects of GLUT1 on cellular hypertrophy and injury could also promote changes of diabetic nephropathy. Strategies to prevent GLUT1 overexpression could ameliorate or prevent the progression of diabetic nephropathy.

    Title Mouse Models of Diabetic Nephropathy.
    Date June 2005
    Journal Journal of the American Society of Nephrology : Jasn
    Excerpt

    Mice provide an experimental model of unparalleled flexibility for studying mammalian diseases. Inbred strains of mice exhibit substantial differences in their susceptibility to the renal complications of diabetes. Much remains to be established regarding the course of diabetic nephropathy (DN) in mice as well as defining those strains and/or mutants that are most susceptible to renal injury from diabetes. Through the use of the unique genetic reagents available in mice (including knockouts and transgenics), the validation of a mouse model reproducing human DN should significantly facilitate the understanding of the underlying genetic mechanisms that contribute to the development of DN. Establishment of an authentic mouse model of DN will undoubtedly facilitate testing of translational diagnostic and therapeutic interventions in mice before testing in humans.

    Title Induction of Antioxidant Enzymes in Murine Podocytes Precedes Injury by Puromycin Aminonucleoside.
    Date April 2005
    Journal Kidney International
    Excerpt

    An imbalance between the generation of reactive oxygen species (ROS) and antioxidant defense mechanisms has been suggested to play an important role in podocyte injury in nephrotic syndrome. Experimental nephrotic syndrome induced by injection of puromycin aminonucleoside (PAN) into rats is a well-established model of nephrotic syndrome, and can be largely prevented by pretreatment with antioxidant enzymes (AOE), suggesting that podocyte injury may be mediated by ROS.

    Title Effects of Ppar-gamma Ligands on Vascular Smooth Muscle Marker Expression in Hypertensive and Normal Arteries.
    Date March 2005
    Journal American Journal of Physiology. Heart and Circulatory Physiology
    Excerpt

    Having previously demonstrated that glucose transporter-4 (GLUT4) expression was reduced in aortas and carotid arteries of deoxycorticosterone acetate (DOCA) salt-hypertensive rats, we hypothesized that troglitazone (TG), through activation of peroxisome proliferator-activated receptor-gamma (PPAR-gamma), would stabilize GLUT4 expression and possibly preserve the differentiated phenotype in vascular smooth muscle cells. In DOCA salt-hypertensive rats treated with TG (100 mg/day), there was a significant (P < 0.001) decrease in systolic blood pressure (BP; 149.9 +/- 4.4 mmHg) compared with the untreated DOCA salt-hypertensive rats (202.2 +/- 10.34 mmHg). Separate trials with rosiglitazone (RS; 3 mg/day) demonstrated a significant (P < 0.001) decrease in BP (DOCA salt, 164.2 +/- 9.8 vs. DOCA-RS, 124.9 +/- 3.7 mmHg) comparable to that with TG. Expression of GLUT4, h-caldesmon, and smooth muscle myosin heavy chain SM2 was significantly decreased in aortas of DOCA salt-hypertensive rats and was reversed by TG to levels similar to those in aortas of sham-treated rats. TG (50 microM) induced GLUT4 and h-caldesmon expression in 24-h culture of explanted carotid arteries of DOCA salt-hypertensive rats, and the endogenous PPAR-gamma ligand 15-deoxy-Delta(12-14)-prostaglandin J(2) (PGJ(2); 20 microM) and TG (50 microM) similarly increased GLUT4, h-caldesmon, and SM2 protein expression in explanted aortas. The expression of activated, phosphorylated Akt was increased by PGJ(2) and TG with no significant effect on total Akt levels. Inhibition of phosphorylated Akt expression using the phosphatidylinositol 3-kinase inhibitor LY-294002 (16 microM) abrogated the increased expression of h-caldesmon and SM2. These data demonstrate that PPAR-gamma agonists maintain or induce expression of markers of the contractile phenotype independently of their effects on hypertension, and that this effect may be mediated through activation of phosphatidylinositol 3-kinase/Akt.

    Title Glut1-deficient Mice Exhibit Impaired Endothelium-dependent Vascular Relaxation.
    Date January 2005
    Journal European Journal of Pharmacology
    Excerpt

    We tested the hypothesis that decreased glucose transporter 1 (GLUT1) expression alters endothelial function. Nitric oxide-dependent endothelial relaxation, but not endothelium-independent relaxation, was significantly reduced in aortas from transgenic mice expressing GLUT1 antisense mRNA, compared to aortas from nontransgenic littermates. These data suggest that GLUT1-dependent glucose metabolism may play an important role in regulating endothelial function.

    Title Increased Vascular Sensitivity and Connexin43 Expression After Sympathetic Denervation.
    Date September 2004
    Journal Cardiovascular Research
    Excerpt

    Objective: Following denervation, arteries demonstrate a heightened sensitivity to alpha-adrenergic agonists and increased oscillatory contractions that may partly result from increased gap junction expression. Hence, we wanted to study the effect of sympathetic denervation on connexin43 (Cx43) expression and agonist-induced contractility in the vascular smooth muscle (VSM). Methods: Effects of denervation with reserpine (3 mg/kg/day, i.p.) or topical 5% phenol-glycerol on VSM contractions and expression of the gap junction Cx43 mRNA by reverse transcriptase polymerase chain reaction (RT-PCR) and Western blotting for Cx43 protein were examined. Wistar-Kyoto (WKY) rat tail arteries were exposed to norepinephrine (NE) (10(-9)-10(-5) M). Reactivity was also examined in the carotid arteries and thoracic aortas from Cx43 heterozygote deficient (KO) mice. Results: The concentration for NE-induced contraction was lower in reserpine- and phenol-treated vessels than controls (p<0.05). NE-induced oscillatory activity (OA) was seen in 5/5 reserpine- and 5/8 phenol-treated vessels vs. 0/12 controls (p<0.05). Spontaneous OA was observed more frequently in carotid and aortic rings from WT than Cx43 KO rings. Cumulative OA in response to alpha-adrenergic stimulation was significantly greater in WT carotid (429+/-101 vs. 128+/-7 mN s, p<0.05) and aortic rings (337+/-85 vs. 134+/-11 mN s, p<0.05) than in Cx43 KO rings. Following denervation, RT-PCR showed significantly increased levels of Cx43 mRNA (p<0.05). Western blot analysis revealed near doubling of Cx43 protein (p<0.05). Conclusion: We conclude that sympathetic denervation results in increased expression of Cx43, which in turn, contributes to increased spontaneous and agonist-induced OA in VSM.

    Title How Glucose and Glucose Transporters Protect Cardiac Myocytes.
    Date June 2004
    Journal Journal of Molecular and Cellular Cardiology
    Title Glucose Transporter-1-deficient Mice Exhibit Impaired Development and Deformities That Are Similar to Diabetic Embryopathy.
    Date April 2004
    Journal Proceedings of the National Academy of Sciences of the United States of America
    Excerpt

    The hyperglycemia of maternal diabetes suppresses the glucose transporter-1 (GLUT1) facilitative glucose transporter 49-66% in preimplantation embryos. Glucose uptake is reduced and apoptosis is activated. We hypothesized that the reduction of embryonic GLUT1 may play a key role in the malformations of diabetic embryopathy. Therefore, we produced GLUT1-deficient transgenic mice [i.e., antisense-GLUT1 (GT1AS)] to determine whether GLUT1 deficiency alone could reproduce the growth defects. Early cell division of fertilized mouse eggs injected with GT1AS was markedly impaired, P < 0.001 vs. controls. Two populations of preimplantation embryos obtained from GT1AS x GT1AS heterozygote matings exhibited reduction of the 2-deoxyglucose uptake rate: one by 50% (presumed heterozygotes, P < 0.001 vs. control) and the other by 95% (presumed homozygotes, P < 0.001 vs. heterozygotes). Embryonic GLUT1 deficiency in the range reported with maternal diabetes was associated with growth retardation and developmental malformations similar to those described in diabetes-exposed embryos: intrauterine growth retardation (31.1%), caudal regression (9.8%), anencephaly with absence of the head (6.6%), microphthalmia (4.9%), and micrognathia (1.6%). Reduced body weight (small embryos, <70% of the nontransgenic body weight) was accompanied by other malformations and a 56% reduction of GLUT1 protein, P < 0.001 vs. nonsmall embryos (body weight >or=70% normal). The heart, brain, and kidneys of embryonic day 18.5 GT1AS embryos exhibited 24-51% reductions of GLUT1 protein. The homozygous GT1AS genotype was lethal during gestation. Reduced embryonic GLUT1 was associated with the appearance of apoptosis. Therefore, GLUT1 deficiency may play a role in producing embryonic malformations resulting from the hyperglycemia of maternal diabetes. Late gestational macrosomia was absent, apparently requiring a different mechanism.

    Title Trophic Factors and Cytokines in Early Diabetic Glomerulopathy.
    Date January 2004
    Journal Experimental Diabesity Research
    Excerpt

    The intent of this review is to focus on recent advances in the understanding of the factors responsible for the progressive pathologic features of diabetic kidney disease, with special attention to various growth factors and cytokines that appear to be important in this process. In addition, emphasis is centered on relatively early stages of the disease, because animal models have been most helpful to date in understanding this stage of the disease process. Although tubulointerstitial changes are of critical importance in the progression of diabetic nephropathy, especially in the evolution to end-stage renal disease, there is a general consensus that glomerular pathology occurs first. Therefore, attention is limited to factors that may be important in the development of early diabetic glomerulopathy, including transforming growth factor-beta (TGF-beta), insulin-like growth factor (IGF)-I, vascular endothelial growth factor (VEGF)-A, and connective tissue growth factor (CTGF).

    Title Inability of Serotonin to Activate the C-jun N-terminal Kinase and P38 Kinase Pathways in Rat Aortic Vascular Smooth Muscle Cells.
    Date January 2004
    Journal Bmc Pharmacology
    Excerpt

    Serotonin (5-HT, 5-hydroxytryptamine) activates the Extracellular Signal-Regulated Kinase (ERK)/ Mitogen-Activated Protein Kinase (MAPK) pathways, in vascular smooth muscle cells. Parallel MAPK pathways, the c-Jun N-terminal Kinase (JNK) and p38 pathway, are activated by stimulators of the ERK/MAPK pathway. We hypothesized that 5-HT would activate the JNK and p38 pathways in rat vascular smooth muscle cells.

    Title Targeted Disruption of the Pept2 Gene Markedly Reduces Dipeptide Uptake in Choroid Plexus.
    Date April 2003
    Journal The Journal of Biological Chemistry
    Excerpt

    The presence of multiple oligopeptide transporters in brain has generated considerable interest as to their physiological role in neuropeptide homeostasis, pharmacologic importance, and potential as a target for drug delivery through the blood-brain and blood-cerebrospinal fluid barriers. To understand further the purpose of specific peptide transporters in brain, we have generated PEPT2-deficient mice by targeted gene disruption. Homozygous PepT2 null mice lacked expression of PEPT2 mRNA and protein in choroid plexus and kidney, tissues in which PepT2 is normally expressed, whereas heterozygous mice displayed PepT2 expression levels that were intermediate between those of wild-type and homozygous null animals. Mutant PepT2 null mice were found to be viable, grew to normal size and weight, and were without obvious kidney or brain abnormalities. Notwithstanding the lack of apparent biological effects, the proton-stimulated uptake of 1.9 microm glycylsarcosine (a model, hydrolysis-resistant dipeptide) in isolated choroid plexus was essentially ablated (i.e. residual activity of 10.9 and 3.9% at 5 and 30 min, respectively). These novel findings provide strong evidence that, under the experimental conditions of this study, PEPT2 is the primary member of the peptide transporter family responsible for dipeptide uptake in choroid plexus tissue.

    Title Pi3-kinase-induced Hyperreactivity in Doca-salt Hypertension is Independent of Gsk-3 Activity.
    Date April 2003
    Journal Hypertension
    Excerpt

    Phosphatidylinositol 3-kinase (PI3K) activity is increased in aortae from deoxycorticosterone (DOCA)-salt rats and enhanced PI3K activity contributes to the arterial hyperreactivity in these animals. Because PI3K activity is increased in DOCA-salt hypertension, we postulated that phosphorylation of Akt and glycogen synthase kinase 3 (GSK-3), serine threonine kinases that are downstream of PI3K, would be increased in DOCA-salt hypertension. In this study, we focused on GSK-3. Because GSK-3 activity is reduced by phosphorylation, we expected that its activity would be reduced in DOCA-salt hypertensive arteries and that reduced GSK-3 activity could contribute to enhanced adrenergic signaling and vascular smooth muscle hypertrophy that augment the heightened contractile response in DOCA-salt hypertension. Surprisingly, we observed a decrease in phosphorylation of GSK-3, indicating an increase in GSK-3 activity. To determine whether increased GSK-3 activity contributes to altered arterial reactivity in DOCA-salt animals, we measured isometric contraction to norepinephrine (NE) in the presence and absence of PI3K or GSK-3 inhibition. Addition of LY294002 (20 micromol/L), a PI3K inhibitor, resulted in a rightward shift in response to NE and normalized the NE-induced contractions in the DOCA hypertensive vessels. SB415286, a GSK-3 inhibitor, resulted in a slight rightward shift in response to NE in the DOCA-salt vessels. Thus, enhanced GSK-3 activity modestly augments the effects of PI3K but does not appear to contribute greatly to the altered arterial reactivity in DOCA-salt hypertension.

    Title Evidence for a Novel Tgf-beta1-independent Mechanism of Fibronectin Production in Mesangial Cells Overexpressing Glucose Transporters.
    Date April 2003
    Journal Diabetes
    Excerpt

    Recent experimental work indicates that the hyperglycemia-induced increase in mesangial matrix production, which is a hallmark in the development of diabetic nephropathy, is mediated by increased expression of GLUT1. Mesangial cells stably transfected with human GLUT1 mimic the effect of hyperglycemia on the production of the extracellular matrix proteins, particularly fibronectin, when cultured under normoglycemic conditions. Our investigation of the molecular mechanism of this effect has revealed that the enhanced fibronectin production was not mediated by the prosclerotic cytokine transforming growth factor (TGF)-beta1. We found markedly increased nuclear content in Jun proteins, leading to enhanced DNA-binding activity of activating protein 1 (AP-1). AP-1 inhibition reduced fibronectin production in a dosage-dependent manner. Moreover, inhibition of classic protein kinase C (PKC) isoforms prevented both the activation of AP-1 and the enhanced fibronectin production. In contrast to mesangial cells exposed to high glucose, no activation of the hexosamine biosynthetic, p38, or extracellular signal-related kinase 1 and 2 mitogen-activated protein kinase pathways nor any increase in TGF-beta1 synthesis could be detected, which could be explained by the absence of oxidative stress in cells transfected with the human GLUT1 gene. Our data indicate that increased glucose uptake and metabolism induce PKC-dependent AP-1 activation that is sufficient for enhanced fibronectin production, but not for increased TGF-beta1 expression.

    Title Glucose Uptake and Adenoviral Mediated Glut1 Infection Decrease Hypoxia-induced Hif-1alpha Levels in Cardiac Myocytes.
    Date March 2003
    Journal Journal of Molecular and Cellular Cardiology
    Excerpt

    Hypoxia causes a large array of adaptive and physiological responses in all cells including cardiac myocytes. In order to elucidate the molecular effects of increased glucose flux on hypoxic cardiac myocytes we focused on the basic helix-loop-helix transcription factor, hypoxia inducible factor 1 alpha (HIF-1alpha), which is rapidly upregulated in hypoxic cells and elicits a number of responses including augmentation of glucose uptake. Primary cultures of neonatal rat cardiac myocytes as well as embryonic rat heart-derived myogenic H9c2 cells demonstrated a significant upregulation of HIF-1alpha when subjected to hypoxia of 6-8h in the absence of glucose. Re-addition of extracellular glucose to the medium resulted in a decrease of HIF-1alpha levels by almost 50%. This glucose effect was blocked by addition of glycolytic inhibitors. In addition, glucose uptake and glycolysis resulted in substantial decreased levels of p53, which is regulated by HIF-1alpha. Adenoviral infection of cultures of cardiac myocytes with the facilitative glucose transporter, GLUT1 followed by hypoxia of 24h also resulted in a significant reduction in the protein expression of HIF-1alpha compared to control vector-infected cultures. GLUT1 infected cultures also demonstrated fewer apoptotic cells and a reduction in the release of cytochrome c after hypoxia. Inhibition of the ubiquitin-proteasomal pathway by a variety of 26S proteasomal inhibitors increased HIF-1alpha to similar levels under both normoxic and hypoxic conditions and in the presence or absence of glucose. This result suggested that glucose induces HIF-1alpha degradation via a proteasomal pathway. This conclusion was substantiated by immunoprecipitation experiments of total cell extracts, which demonstrated an increase of ubiquitinated HIF-1alpha relative to total HIF-1alpha in the presence of glucose during hypoxia. Thus, glucose as well as GLUT1 overexpression diminishes hypoxia-induced HIF-1alpha protein via an ubiquitin-proteasomal pathway in hypoxic cardiac myocytes. This represents a novel feedback mechanism that may play an important role in adaptation of cardiac myocytes to hypoxia and ischemia.

    Title Enhanced Glycogen Synthase Kinase-3beta Activity Mediates Hypoxia-induced Apoptosis of Vascular Smooth Muscle Cells and is Prevented by Glucose Transport and Metabolism.
    Date January 2003
    Journal The Journal of Biological Chemistry
    Excerpt

    Hypoxia triggers apoptosis in a number of different cell types largely through a mitochondrial cell death pathway, which can be abrogated for the most part by enhanced glucose metabolism. The purpose of the current study was to identify intracellular signaling mechanisms that mediate hypoxia-induced apoptosis and are regulated by glucose metabolism. Hypoxia-induced apoptosis in vascular smooth muscle cells and COS-7 cells was accompanied by a significant reduction in Akt and glycogen synthase kinase-3 (GSK-3) phosphorylation resulting in increased GSK-3 activity. Morphologic features of apoptosis, as well as caspases 3 and 9 activation, were prevented by GSK-3 inhibition with either LiCl or SB216763. Phosphorylation of Akt and GSK-3 was enhanced by glucose metabolism or overexpression of the glucose transporter, GLUT1, and was prevented by glycolytic inhibition. These findings indicate that GSK-3 is an important mediator of hypoxia-induced apoptosis and that GSK-3-mediated apoptotic effects occur via activation of the mitochondrial death pathway. Moreover, the results suggest that prevention of hypoxia-mediated apoptosis by enhanced glucose transport and metabolism results, in part, from inhibition of GSK-3 activation.

    Title Decreased Vascular Glucose Transporter Expression and Glucose Uptake in Doca-salt Hypertension.
    Date December 2001
    Journal Journal of Hypertension
    Excerpt

    Because glucose uptake and metabolism can affect vascular smooth muscle cell function, we proposed that animals with hypertension might develop alterations in glucose transporter expression in vascular smooth muscle cells that were responsible for some of the vascular abnormalities characteristic of hypertension.

    Title Hypoxia Induces Apoptosis Via Two Independent Pathways in Jurkat Cells: Differential Regulation by Glucose.
    Date December 2001
    Journal American Journal of Physiology. Cell Physiology
    Excerpt

    Glucose uptake and metabolism inhibit hypoxia-induced apoptosis in a variety of cell types, but the underlying molecular mechanisms remain poorly understood. In the present study, we explore hypoxia-mediated cell death pathways in Jurkat cells in the presence and absence of extracellular glucose. In the absence of extracellular glucose, hypoxia caused cytochrome c release, caspase 3 and poly(ADP-ribose)polymerase cleavage, and DNA fragmentation; this apoptotic response was blocked by the caspase 9 inhibitor z-LEHD-FMK. The presence of extracellular glucose during hypoxia prevented cytochrome c release and activation of caspase 9 but did not prevent apoptosis in Jurkat cells. In these conditions, overexpression of the caspase 8 inhibitor v-FLIP prevented hypoxia-mediated cell death. Thus hypoxia can stimulate two apoptotic pathways in Jurkat cells, one dependent on cytochrome c release from mitochondria that is prevented by glucose uptake and metabolism, and the other independent of cytochrome c release and resulting from activation of the death receptor pathway, which is accelerated by glucose uptake and metabolism.

    Title Developmental Expression of Pept1 and Pept2 in Rat Small Intestine, Colon, and Kidney.
    Date August 2001
    Journal Pediatric Research
    Excerpt

    Mammalian peptide transporters (PEPT1 and PEPT2) play a pivotal role in the absorption of small peptides from the intestine and kidney, respectively, and in the disposition and targeting of peptide or mimetic drugs. However, there are few reports on the molecular basis of their regulation, especially in the young. The aim of this study was to determine the developmental expression of intestinal and renal oligopeptide transporters in rats from embryonic to adult ages. Intestinal segments were collected (i.e. duodenum, jejunum, ileum, and colon) along with whole kidney, and their mRNA and protein levels were measured. Expression levels of PEPT1 were maximal 3-5 d after birth in the duodenum, jejunum, and ileum, and then declined rapidly. Expression was increased transiently at d 24, most notably in the ileum. Adult protein levels were approximately 70% of that observed on d 3-5. Significant PEPT1 expression was observed in colon during the first week of life, but levels were undetectable shortly thereafter through adulthood. PEPT1 and PEPT2 expression is less regulated in rat kidney and more pronounced in older animals. Peptide transporters were also present as early as d 20 of fetal life for all tissues tested. These results are unique in providing the developmental expression of peptide transporter mRNA and protein in distinct regions of the small intestine, colon, and kidney in rat. Our findings suggest that intestinal expression of PEPT1 is induced postpartum, possibly by suckling, and again at the time of weaning, and that the colon may participate in peptide transport early in life.

    Title Characterization of Sodium Channel Alpha- and Beta-subunits in Rat and Mouse Cardiac Myocytes.
    Date May 2001
    Journal Circulation
    Excerpt

    Sodium channels isolated from mammalian brain are composed of alpha-, beta(1)-, and beta(2)-subunits. The composition of sodium channels in cardiac muscle, however, has not been defined, and disagreement exists over which beta-subunits are expressed in the myocytes. Some investigators have demonstrated beta(1) expression in heart. Others have not detected any auxiliary subunits. On the basis of Northern blot analysis of total RNA, beta(2) expression has been thought to be exclusive to neurons and absent from cardiac muscle.

    Title Antisense Glut-1 Protects Mesangial Cells from Glucose Induction of Glut-1 and Fibronectin Expression.
    Date April 2001
    Journal American Journal of Physiology. Renal Physiology
    Excerpt

    A stable clone of rat mesangial cells expressing antisense GLUT-1 (i.e., MCGT1AS cells) was developed to protect them from high glucose exposure. GLUT-1 protein was reduced 50%, and the 2-deoxy-[(3)H]glucose uptake rate was reduced 33% in MCGT1AS. MCLacZ control cells and MCGT1 GLUT-1-overexpressing cells were used for comparisons. In MCLacZ, 20 mM D-glucose increased GLUT-1 transcription 90% vs. no increase in MCGT1AS. Glucose (8 mM) and 12 mM xylitol [a hexose monophosphate (HMP) shunt substrate] did not stimulate GLUT-1 transcription. An 87% replacement of the standard 8 mM D-glucose with 3-O-methylglucose reduced GLUT-1 transcription 80%. D-Glucose (20 mM) increased fibronectin mRNA and protein by 47 and 100%, respectively, in MCLacZ vs. no increases in MCGT1AS. Fibronectin synthesis was elevated 48% in MCGT1 and reduced 44% in MCGT1AS. We conclude that 1) transcription of GLUT-1 in response to D-glucose depends on glucose metabolism, although not through the HMP shunt, and 2) antisense GLUT-1 treatment of mesangial cells blocks D-glucose-induced GLUT-1 and fibronectin expression, thereby demonstrating a protective effect that could be beneficial in the setting of diabetes.

    Title Regulation of Glucose Transport in Cultured Schwann Cells.
    Date September 2000
    Journal Journal of the Peripheral Nervous System : Jpns
    Excerpt

    Glucose is the major source of metabolic energy in the peripheral nerve. Energy derived from glucose is mostly utilized for axonal repolarization. One route by which glucose may reach the axon is by crossing the Schwann cells that initially surround the axons. Considering the ability of neurons to control many glial cell functions, we postulated that Schwann cell glucose transporters might be transiently regulated by axonal contact. Glucose transport was studied in a cultured, differentiated rat Schwann cell line stably expressing SV40 T antigen regulated by a synthetic mouse metallothionein promoter. 3[H]-2-deoxy-D-glucose uptake was measured in cultured cells in basal and in various experimental conditions. Glucose transporter gene expression was determined after RNA isolation from cultured cells through Northern and RNAse protection assay. In vitro, Schwann cells were found to express high-affinity, insulin-insensitive, facilitative glucose transporters and predominantly GLUT1 mRNA. Schwann cell 2-deoxyglucose uptake was increased by axolemmal membranes or forskolin but unchanged by elevated glucose levels. Regulation of Schwann cell glucose transporters by axolemma and their resistance to glucose-induced down-regulation suggest extrinsic rather than intrinsic regulation that might enhance Schwann cell vulnerability to glucotoxicity.

    Title Glut-1 Reduces Hypoxia-induced Apoptosis and Jnk Pathway Activation.
    Date June 2000
    Journal American Journal of Physiology. Endocrinology and Metabolism
    Excerpt

    Many studies have suggested that enhanced glucose uptake protects cells from hypoxic injury. More recently, it has become clear that hypoxia induces apoptosis as well as necrotic cell death. We have previously shown that hypoxia-induced apoptosis can be prevented by glucose uptake and glycolytic metabolism in cardiac myocytes. To test whether increasing the number of glucose transporters on the plasma membrane of cells could elicit a similar protective response, independent of the levels of extracellular glucose, we overexpressed the facilitative glucose transporter GLUT-1 in a vascular smooth muscle cell line. After 4 h of hypoxia, the percentage of cells that showed morphological changes of apoptosis was 30.5 +/- 2.6% in control cells and only 6.0 +/- 1.1 and 3.9 +/- 0.3% in GLUT-1-overexpressing cells. Similar protection against cell death and apoptosis was seen in GLUT-1-overexpressing cells treated for 6 h with the electron transport inhibitor rotenone. In addition, hypoxia and rotenone stimulated c-Jun-NH(2)-terminal kinase (JNK) activity >10-fold in control cell lines, and this activation was markedly reduced in GLUT-1-overexpressing cell lines. A catalytically inactive mutant of MEKK1, an upstream kinase in the JNK pathway, reduced hypoxia-induced apoptosis by 39%. These findings show that GLUT-1 overexpression prevents hypoxia-induced apoptosis possibly via inhibition of stress-activated protein kinase pathway activation.

    Title Expression of Peroxisomal Proliferator-activated Receptors and Retinoid X Receptors in the Kidney.
    Date January 2000
    Journal The American Journal of Physiology
    Excerpt

    The discovery that 15-deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2) is a ligand for the gamma-isoform of peroxisome proliferator-activated receptor (PPAR) suggests nuclear signaling by prostaglandins. Studies were undertaken to determine the nephron localization of PPAR isoforms and their heterodimer partners, retinoid X receptors (RXR), and to evaluate the function of this system in the kidney. PPARalpha mRNA, determined by RT-PCR, was found predominately in cortex and further localized to proximal convoluted tubule (PCT); PPARgamma was abundant in renal inner medulla, localized to inner medullary collecting duct (IMCD) and renal medullary interstitial cells (RMIC); PPARbeta, the ubiquitous form of PPAR, was abundant in all nephron segments examined. RXRalpha was localized to PCT and IMCD, whereas RXRbeta was expressed in almost all nephron segments examined. mRNA expression of acyl-CoA synthase (ACS), a known PPAR target gene, was stimulated in renal cortex of rats fed with fenofibrate, but the expression was not significantly altered in either cortex or inner medulla of rats fed with troglitazone. In cultured RMIC cells, both troglitazone and 15d-PGJ2 significantly inhibited cell proliferation and dramatically altered cell shape by induction of cell process formation. We conclude that PPAR and RXR isoforms are expressed in a nephron segment-specific manner, suggesting distinct functions, with PPARalpha being involved in energy metabolism through regulating ACS in PCT and with PPARgamma being involved in modulating RMIC growth and differentiation.

    Title Arc Inhibits Cytochrome C Release from Mitochondria and Protects Against Hypoxia-induced Apoptosis in Heart-derived H9c2 Cells.
    Date December 1999
    Journal Circulation Research
    Excerpt

    Ischemia induces apoptosis as well as necrosis of cardiac myocytes. We recently reported the cloning of a cDNA that encodes an apoptotic inhibitor, ARC, that is expressed predominantly in cardiac and skeletal muscle. In the present study, we examined the ability of ARC to protect rat embryonic heart-derived H9c2 cells from apoptosis induced by hypoxia, a component of ischemia. We found that H9c2 cells express ARC and that exposure to hypoxia substantially reduces ARC expression while inducing apoptosis. Transfected H9c2 cells in which cytosolic ARC protein levels remain elevated during hypoxia were significantly more resistant to hypoxia-induced apoptosis than parental H9c2 cells or H9c2 cells transfected with a control vector. Loss of endogenous ARC in the cytosol of H9c2 cells was associated with translocation of ARC from the cytosol to intracellular membranes, release of cytochrome c from the mitochondria, activation of caspase-3, poly(ADP-ribose)polymerase (PARP) cleavage, and DNA fragmentation. All of these events were inhibited in H9c2 cells overexpressing ARC when compared with control cells. In contrast, caspase inhibitors prevented PARP cleavage but not cytochrome c release, suggesting that exogenously expressed ARC acts upstream of caspase activation in this model of apoptosis. These results demonstrate that ARC can protect heart myogenic H9c2 cells from hypoxia-induced apoptosis, and that ARC prevents cytochrome c release by acting upstream of caspase activation, perhaps at the mitochondrial level.

    Title Glucose Transporters Control Gene Expression of Aldose Reductase, Pkcalpha, and Glut1 in Mesangial Cells in Vitro.
    Date August 1999
    Journal The American Journal of Physiology
    Excerpt

    The process linking increased glucose utilization and activation of metabolic pathways leading to end-organ damage from diabetes is not known. We have previously described rat mesangial cells that were transduced to constitutively express the facilitative glucose transporter 1 (GLUT1, MCGT1 cells) or bacterial beta-galactosidase (MCLacZ, control cells). Glucose transport was rate limiting for extracellular matrix production in the MCGT1 cells. In the present work, we investigated the effect of GLUT1 overexpression in mesangial cells on aldose reductase (AR), protein kinase Calpha (PKCalpha), and native GLUT1 transcript levels, to determine whether changes in GLUT1 alone could regulate their expression in the absence of high extracellular glucose concentrations. MCGT1 cells grown in normal (8 mM) or elevated (20 mM) glucose had elevated abundance of AR, PKCalpha, and the native GLUT1 transcripts compared with control cells. AR protein levels, AR activity, sorbitol production, and PKCalpha protein content were also greater in the MCGT1 cells than in control cells grown in the same media. This is the first report of the concomitant activation of AR, PKCalpha, and GLUT1 genes by enhanced GLUT1 expression. We conclude that increased GLUT1 expression leads to a positive feedback of greater GLUT1 expression, increased AR expression and activity with polyol accumulation, and increased total and active PKCalpha protein levels, which leads to detrimental stimulation of matrix protein synthesis by diabetic mesangial cells.

    Title Mechanical Stretch and Angiotensin Ii Differentially Upregulate the Renin-angiotensin System in Cardiac Myocytes In Vitro.
    Date August 1999
    Journal Circulation Research
    Excerpt

    Pressure overload in vivo results in left ventricular hypertrophy and activation of the renin-angiotensin system in the heart. Mechanical stretch of neonatal rat cardiac myocytes in vitro causes secretion of angiotensin II (Ang II), which in turn plays a pivotal role in mechanical stretch-induced hypertrophy. Although in vivo data suggest that the stimulus of hemodynamic overload serves as an important modulator of cardiac renin-angiotensin system (RAS) activity, it is not clear whether observed upregulation of RAS genes is a direct effect of hemodynamic stress or is secondary to neurohumoral effects in response to hemodynamic overload. Moreover, it is unclear whether activation of the local RAS in response to hemodynamic overload predominantly occurs in cardiac myocytes or fibroblasts or both. In the present study, we examined the effect of mechanical stretch on expression of angiotensinogen, renin, angiotensin-converting enzyme (ACE), and Ang II receptor (AT(1A), AT(1B), and AT(2)) genes in neonatal rat cardiac myocytes and cardiac fibroblasts in vitro. The level of expression of angiotensinogen, renin, ACE, and AT(1A) genes was low in unstretched cardiac myocytes, but stretch upregulated expression of these genes at 8 to 24 hours. Stimulation of cardiac myocytes with Ang II also upregulated expression of angiotensinogen, renin, and ACE genes, whereas it downregulated AT(1A) and did not affect AT(1B) gene expression. Although losartan, a specific AT(1) antagonist, completely inhibited Ang II-induced upregulation of angiotensinogen, renin, and ACE genes, as well as stretch-induced upregulation of AT(1A) expression, it did not block upregulation of angiotensinogen, renin, and ACE genes by stretch. Western blot analyses showed increased expression of angiotensinogen and renin protein at 16 to 24 hours of stretch. The ACE-like activity was also significantly elevated at 24 hours after stretch. Radioligand binding assays revealed that stretch significantly upregulated the AT(1) density on cardiac myocytes. Interestingly, stretch of cardiac fibroblasts did not result in any discernible increases in the expression of RAS genes. Our results indicate that mechanical stretch in vitro upregulates both mRNA and protein expression of RAS components specifically in cardiac myocytes. Furthermore, components of the cardiac RAS are independently and differentially regulated by mechanical stretch and Ang II in neonatal rat cardiac myocytes.

    Title Glucose Uptake and Glycolysis Reduce Hypoxia-induced Apoptosis in Cultured Neonatal Rat Cardiac Myocytes.
    Date June 1999
    Journal The Journal of Biological Chemistry
    Excerpt

    Myocardial ischemia/reperfusion is well recognized as a major cause of apoptotic or necrotic cell death. Neonatal rat cardiac myocytes are intrinsically resistant to hypoxia-induced apoptosis, suggesting a protective role of energy-generating substrates. In the present report, a model of sustained hypoxia of primary cultures of Percoll-enriched neonatal rat cardiac myocytes was used to study specifically the modulatory role of extracellular glucose and other intermediary substrates of energy metabolism (pyruvate, lactate, propionate) as well as glycolytic inhibitors (2-deoxyglucose and iodoacetate) on the induction and maintenance of apoptosis. In the absence of glucose and other substrates, hypoxia (5% CO2 and 95% N2) caused apoptosis in 14% of cardiac myocytes at 3 h and in 22% of cells at 6-8 h of hypoxia, as revealed by sarcolemmal membrane blebbing, nuclear fragmentation, and chromatin condensation (Hoechst staining), terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining, and DNA laddering. This was accompanied by translocation of cytochrome c from the mitochondria to the cytosol and cleavage of the death substrate poly(ADP-ribose) polymerase. Cleavage of poly(ADP-ribose) polymerase and DNA laddering were prevented by preincubation with the caspase inhibitors benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (zVAD-fmk) and benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethyl ketone (zDEVD-fmk), indicating activation of caspases in the apoptotic process. The caspase inhibitor zDEVD-fmk also partially inhibited cytochrome c translocation. The presence of as little as 1 mM glucose, but not pyruvate, lactate, or propionate, before hypoxia prevented apoptosis. Inhibiting glycolysis by 2-deoxyglucose or iodoacetate, in the presence of glucose, reversed the protective effect of glucose. This study demonstrates that glycolysis of extracellular glucose, and not other metabolic pathways, protects cardiac myocytes from hypoxic injury and subsequent apoptosis.

    Title Localization of Pept1 and Pept2 Proton-coupled Oligopeptide Transporter Mrna and Protein in Rat Kidney.
    Date June 1999
    Journal The American Journal of Physiology
    Excerpt

    To determine the renal localization of oligopeptide transporters, Northern blot analyses were performed and polyclonal antisera were generated against PEPT1 and PEPT2, the two cloned rat H+/peptide transporters. Under high-stringency conditions, a 3.0-kb mRNA transcript of rat PEPT1 was expressed primarily in superficial cortex, whereas a 3.5-kb mRNA transcript of PEPT2 was expressed primarily in deep cortex/outer stripe of outer medulla. PEPT1 antisera detected a specific band on immunoblots of renal and intestinal brush-border membrane vesicles (BBMV) with an apparent mobility of approximately 90 kDa. PEPT2 antisera detected a specific broad band of approximately 85 kDa in renal but not in intestinal BBMV. PEPT1 immunolocalization experiments showed detection of a brush border antigen in S1 segments of the proximal tubule and in the brush border of villi from all segments of the small intestine. In contrast, PEPT2 immunolocalization was primarily confined to the brush border of S3 segments of the proximal tubule. All other nephron segments in rat were negative for PEPT1 and PEPT2 staining. Overall, our results conclusively demonstrate that although PEPT1 is expressed in early regions of the proximal tubule (pars convoluta), PEPT2 is specific for the latter regions of proximal tubule (pars recta).

    Title Choroid Plexus Ion Transporter Expression and Cerebrospinal Fluid Secretion.
    Date February 1998
    Journal Acta Neurochirurgica. Supplement
    Excerpt

    The Cl-/HCO3- exchanger (AE2 isoform) and the Na+/K(+)-ATPase at the choroid plexus are both thought to be involved in CSF secretion. However, both transport mechanisms are also postulated to have a role in CSF ion homeostasis raising questions as to which parameters control the expression of these transporters? Northern blots have been used to assess AE2 mRNA levels in rats subjected to alterations in blood pH or blood osmolality (a factor affecting CSF secretion). Six hours of alkalosis induced a 40% increase in AE2 mRNA (p < 0.01), suggesting that alterations in the expression of this transporter play a role in CSF pH homeostasis. In contrast, changes in osmolality did not affect AE2 mRNA. Western blots of Na+/K(+)-ATPase subunits were also examined to determine whether hypo and hyperkalemia affect protein levels of this transporter. There was a positive correlation between the plasma K+ concentration and both alpha 1- and beta 1 subunit protein levels suggesting a role for this transporter in CSF K+ homeostasis. As changes in plasma K+ and pH affect choroid plexus ion transporters but do not appear to alter CSF production, these results suggest the presence of compensatory mechanisms. Understanding of such mechanisms may facilitate therapeutic control of CSF production.

    Title Ae Anion Exchanger Mrna and Protein Expression in Vascular Smooth Muscle Cells, Aorta, and Renal Microvessels.
    Date February 1998
    Journal The American Journal of Physiology
    Excerpt

    Intracellular pH (pHi) is an important regulator of vascular smooth muscle cell (VSMC) tone, contractility, and intracellular Ca2+ concentration. Among the multiple transport processes that regulate VSMC pHi, Na(+)-independent Cl-/HCO3- exchange is the major process that acidifies VSMCs in response to an alkaline load. Here, we characterize, in native and cultured VSMCs, the expression of the AE family of band 3-related anion exchangers, the best studied of these Cl-/HCO3- exchangers. A 4.2-kb AE2 mRNA was present in aorta and in all cultured VSMCs tested. Cultured VSMCs and aorta both expressed a approximately 165-kDa AE2 polypeptide, but a approximately 115-kDa polypeptide was the major AE2-related protein in aorta. AE3 mRNA levels in VSMCs and in arterial tissue were significantly lower than those for AE2, but AE3 or related polypeptides were readily detected by immunoblot and immunolocalization experiments. The approximately 125-kDa AE3 polypeptide was present in an immortalized aortic VSMC line, but the predominant AE3 epitope in aorta and most cultured cells was associated with a polypeptide of M(r) approximately 80 kDa. These data demonstrate the expression in native arteries and in VSMCs of products of the AE2 and AE3 genes, which may contribute to Na(+)-independent Cl-/HCO3- exchange activity in these tissues and cells.

    Title Functional Consequences of Mutations in the Transmembrane Domain and the Carboxy-terminus of the Murine Ae1 Anion Exchanger.
    Date December 1997
    Journal Biochimica Et Biophysica Acta
    Excerpt

    We have characterized mouse AE1-mediated 36Cl- influx and surface AE1 polypeptide expression in Xenopus oocytes injected with cRNA encoding two classes of loss-of-function mutants. The first arose spontaneously. Chimeric mutants constructed with a functional AE1 cDNA localized the site of spontaneous mutation to the transmembrane domain, and DNA sequencing revealed two missense mutations encoding the double-mutant polypeptide V728F/M7301. Each mutation individually produced only partial loss of AE1 transport activity, and coexpression of the individual mutants did not restore full activity. The functional changes produced by the mutations correlated with reduced fractional accumulation of polypeptides at the oocyte surface. The V728F/M7301 polypeptide expressed in mammalian cells displayed complete endoH resistance and rapid degradation. We also examined the effect on AE1 function of engineered removal of its hydrophilic carboxy-terminus. Both delta(c)890 and the internal deletion delta(c)890-917 were functionally inactive in Xenopus oocytes. Lack of transport activity correlated with lack of detectable polypeptide accumulation at the oocyte surface. Coexpression with wt AE1 of some, but not all, of these AE1 mutants partially suppressed wt AE1-mediated 36Cl- uptake. In contrast, coexpression with wt AE1 of soluble N-terminal AE1 fragments was not inhibitory.

    Title Effects of Wortmannin on Insulin- and Ischemia-induced Stimulation of Glut4 Translocation and Fdg Uptake in Perfused Rat Hearts.
    Date November 1997
    Journal Cardiovascular Research
    Excerpt

    Myocardial glucose transport is enhanced by hormonal and other stimuli such as ischemia and hypoxia which induce glucose transporter 4 (GLUT4) translocation. Whether insulin and ischemia share a common signaling mechanism is not yet known. This study investigated whether phosphatidylinositol 3-kinase (PI3K), a signaling intermediate of the insulin-responsible pathway, also participates in the ischemia-induced stimulation of glucose.

    Title Glucose Transporters of the Glomerulus and the Implications for Diabetic Nephropathy.
    Date October 1997
    Journal Kidney International. Supplement
    Excerpt

    Several glucose transporters have recently been identified in glomeruli, and in cultured glomerular cells. These include the facilitative glucose transporter isoforms GLUTs 1, 3 and 4, and sodium-glucose cotransport activity with characteristics of SGLT1. GLUTs 1, 3 and 4 are all high affinity, low capacity, facilitative glucose transporters which typically would be saturated at or near physiologic glucose concentrations. The SGLT transporter of mesangial cells is also a high affinity transporter which similarly could be saturated under normal glucose conditions. This suggests that in order for mesangial cells to take up excessive quantities of glucose in diabetes, changes in glucose transporter expression, translocation or activity may be required. Accordingly, recent investigations discovered positive-feedback regulation of the mesangial cell GLUT1 transporter by glucose, and a regulatory role for GLUT1 in glucose metabolism and extracellular matrix synthesis. Future investigations of glucose transporters in the pathogenesis of diabetic renal disease will now likely proceed in multiple directions, including but not limited to: (1) examination of their regulation by growth factors implicated in diabetic nephropathy, and the resultant effects on ECM synthesis; (2) determination of the mechanisms by which GLUT1 regulates the expression of aldose reductase, PKC, GLUT1, and other genes in the mesangial cell; and (3) Suppression of glucose transporters in attempts to prevent high glucose-induced diabetic glomerulosclerosis.

    Title Increased Sarcolemmal Glucose Transporter Abundance in Myocardial Ischemia.
    Date September 1997
    Journal The American Journal of Cardiology
    Excerpt

    Many clinical and laboratory studies suggest that an increase in glucose uptake and metabolism by ischemic myocardium helps protect myocardial cells from irreversible injury. We have examined whether increased sarcolemmal abundance of cardiomyocyte glucose transporters plays a role in this adaptive response. We have shown that acute myocardial ischemia in perfused rat hearts results in increased sarcolemmal abundance of the major glucose transporter, GLUT4, by causing translocation of GLUT4 molecules from an intracellular compartment to the sarcolemma. In nonischemic control hearts only 18 +/- 2.8% of GLUT4 molecules were on the sarcolemma whereas in ischemic hearts this increased to 41 +/- 9.3%. Insulin also caused translocation of GLUT4 molecules to the sarcolemma, and resulted in 61 +/- 2.6% of GLUT4 molecules on the sarcolemma. The combination of ischemia and insulin did not result in additive increases in sarcolemmal GLUT4 abundance. In more persistent or chronic ischemia, the other major myocardial glucose transporter, GLUT1, appears to play an important role. The mRNA for this transporter, which is constitutively expressed on cardiomyocyte sarcolemma, was increased 2.0-fold in regions of hibernating myocardium in humans with coronary heart disease as well as in persistently hypoxic rat neonatal cardiomyocytes in primary culture. In neither of these conditions was GLUT4 mRNA expression increased. Thus, acute myocardial ischemia increases sarcolemmal glucose transporter abundance mainly by translocating previously synthesized GLUT4 molecules from an intracellular compartment, whereas more chronic ischemia also increases GLUT1 abundance via enhanced mRNA expression. Increased GLUT1 and GLUT4 abundance may participate in the augmented glucose uptake of ischemic myocardium and therefore may help protect ischemic myocardium from irreversible injury.

    Title Persistent Myocardial Ischemia Increases Glut1 Glucose Transporter Expression in Both Ischemic and Non-ischemic Heart Regions.
    Date August 1997
    Journal Journal of Molecular and Cellular Cardiology
    Excerpt

    Persistently ischemic myocardium exhibits increased glucose uptake which may contribute to the preservation of myocardial function and viability. Little is known about the specific molecular events which are responsible for this increase in uptake. Therefore, we investigated whether myocardial ischemia induces the gene expression of the major cardiac facilitative glucose transporters, GLUT4 and GLUT1. We determined the expression of myocardial glucose transporter mRNAs and polypeptides after 6 h of regional ischemia in a dog model by semi-quantitative Northern blotting and immunoblotting. GLUT1 but not GLUT4 expression was significantly increased in both ischemic and non-ischemic regions from the experimental hearts when compared to surgical control and normal hearts. GLUT1 mRNA expression was increased 3.4-fold and GLUT1 polypeptide expression was increased 1.7-fold in ischemic hearts when compared to normal or surgical-control hearts. There were no significant regional differences in GLUT1 expression in either normal or ischemic hearts. However, there was a tendency for GLUT1 mRNA expression to be highest in the non-ischemic regions from the 6-h ischemia hearts. These findings suggest that myocardial ischemia induces a factor or factors which stimulate GLUT1 expression in non-ischemic as well as ischemic myocardial regions. Increased GLUT1 expression may play a role in augmenting glucose uptake during ischemia.

    Title D-glucose Stimulates Mesangial Cell Glut1 Expression and Basal and Igf-i-sensitive Glucose Uptake in Rat Mesangial Cells: Implications for Diabetic Nephropathy.
    Date June 1997
    Journal Diabetes
    Excerpt

    The complications of diabetes arise in part from abnormally high cellular glucose uptake and metabolism. To determine whether altered glucose transporter expression may be involved in the pathogenesis of diabetic nephropathy, we investigated the effects of elevated extracellular glucose concentrations on facilitative glucose transporter (GLUT) expression in rat mesangial cells. GLUT1 was the only transporter isoform detected. Cells exposed to 20 mmol/l glucose medium for 3 days demonstrated increases in GLUT1 mRNA (134%, P < 0.002), GLUT1 protein (68%, P < 0.02), and V(max) (50%, P < 0.05) for uptake of the glucose analog [3H]2-deoxyglucose (3H2-DOG), when compared to cells chronically adapted to physiologic glucose concentrations (8 mmol/l). The increase in GLUT1 protein was sustained at 3 months, the latest time point tested (77% above control, P < 0.01). In contrast, hypertonic mannitol had no effect on GLUT1 protein levels. Insulin-like growth factor I (IGF-I; 30 ng/ml) increased the uptake of 3H2-DOG by 28% in 8 mmol/l glucose-treated cells (P < 0.05) and by 75% in cells switched to 20 mmol/l glucose for 3 days (P < 0.005). These increases in 3H2-DOG uptake occurred despite a lack of effect of IGF-I on GLUT1 protein levels (P > 0.5 vs. control). Therefore, hyperglycemia and IGF-I treatment both lead to increases in mesangial cell glucose uptake, and hyperglycemia induces increased GLUT1 expression, which can directly lead to the pathological changes of diabetic nephropathy. The effects of high glucose and of IGF-I to stimulate 3H2-DOG uptake also appear to be additive.

    Title Purification of Pcr Products from Agarose Gels for Direct Sequencing.
    Date February 1997
    Journal Methods in Molecular Biology (clifton, N.j.)
    Title Glucose Transporters in Rat Peripheral Nerve: Paranodal Expression of Glut1 and Glut3.
    Date January 1997
    Journal Metabolism: Clinical and Experimental
    Excerpt

    Peripheral nerve depends on glucose oxidation to energize the repolarization of excitable axonal membranes following impulse conduction, hence requiring high-energy demands by the axon at the node of Ranvier. To enter the axon at this site, glucose must be transported from the endoneurial space across Schwann cell plasma membranes and the axolemma. Such transport is likely to be mediated by facilitative glucose transporters. Although immunohistochemical studies of peripheral nerves have detected high levels of the transporter GLUT1 in endoneurial capillaries and perineurium, localization of glucose transporters to Schwann cells or peripheral axons in vivo has not been documented. In this study, we demonstrate that the GLUT1 transporter is expressed in the plasma membrane and cytoplasm of myelinating Schwann cells around the nodes of Ranvier and in the Schmidt-Lanterman incisures, making them potential sites of transcellular glucose transport. No GLUT1 was detected in axonal membranes. GLUT3 mRNA was expressed only at low levels, but GLUT3 polypeptide was barely detected by immunocytochemistry or immunoblotting in peripheral nerve from young adult rats. However, in 13-month-old rats, GLUT3 polypeptide was present in myelinated fibers, endoneurial capillaries, and perineurium. In myelinated fibers, GLUT3 appeared to be preferentially expressed in the paranodal regions of Schwann cells and nodal axons, but was also present in the internodal aspects of these structures. The results of the present study suggest that both Schwann cell GLUT1 and axonal and Schwann cell GLUT3 are involved in the transport of glucose into the metabolically active regions of peripheral axons.

    Title Chromosomal Mapping of the Rat Slc4a Family of Anion Exchanger Genes, Ae1, Ae2, and Ae3.
    Date October 1996
    Journal Mammalian Genome : Official Journal of the International Mammalian Genome Society
    Title Sa Gene Expression in the Proximal Tubule of Normotensive and Hypertensive Rats.
    Date June 1996
    Journal Hypertension
    Excerpt

    Previous studies have shown that the SA gene is expressed at higher levels in the kidney of genetically hypertensive rats than in control strains and that in hybrid crosses of genetically hypertensive rats and normotensive controls, markers in or close to the SA gene cosegregate with blood pressure. The present studies examine the localization of the SA gene product in the kidney by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR). cDNA was prepared from microdissected nephron segments from Sprague-Dawley (SD) rats, spontaneously hypertensive rats (SHRs), and Wistar-Kyoto (WKY) rats, and RT-PCR was performed using specific primers. In all three strains, SA gene mRNA was found to be abundantly expressed in proximal tubules. SA PCR product was occasionally detected at approximately 100-fold lower abundance in glomeruli, while no signal was obtained from the collecting duct, thick ascending limb of the loop of Henle, or arcuate artery. Within the proximal tubule of normotensive rats, distribution of SA mRNA was found to be strain dependent: in SD rats it was expressed at high levels in the proximal convoluted tubule, whereas in WKY rats it was restricted to the proximal straight tubule. In SHRs, SA PCR product was detected along the entire proximal tubule. Induction of hypertension by renal artery clamping (two-kidney, one-clamp Goldblatt model) did not alter the pattern of expression observed in the SD rat. These results indicate that an extension of SA gene expression to the full length of the proximal tubule accompanies spontaneous hypertension and that in nonhypertensive animals the pattern of gene product expression is more restricted but shows substantial strain variability.

    Title Regional and Segmental Localization of Ae2 Anion Exchanger Mrna and Protein in Rat Kidney.
    Date December 1995
    Journal The American Journal of Physiology
    Excerpt

    Chloride/base exchange activity has been detected in every mammalian nephron segment in which it has been sought. However, in contrast to the Cl-/HCO3- exchanger AE1 in type A intercalated cells, localization of AE2 within the kidney has not been reported. We therefore studied AE2 expression in rat kidney. AE2 mRNA was present in cortex, outer medulla, and inner medulla. Semiquantitative polymerase chain reaction of cDNA from microdissected tubules revealed AE2 cDNA levels as follows [copies of cDNA derived per mm tubule (+/- SE)]: proximal convoluted tubule, 688 +/- 161; proximal straight tubule, 652 +/- 189; medullary thick ascending limb, 1,378 +/- 226; cortical thick ascending limb, 741 +/- 24; cortical collecting duct, 909 +/- 71; and outer medullary collecting duct, 579 +/- 132. AE2 cDNA was also amplified in thin limbs and in inner medullary collecting duct. AE2 polypeptide was detected in all kidney regions. AE2 mRNA and protein were also detected in several renal cell lines. The data are compatible with the postulated roles of AE2 in maintenance of intracellular pH and chloride concentration and with its possible participation in transepithelial transport.

    Title Altered Renal Expression of the Insulin-responsive Glucose Transporter Glut4 in Experimental Diabetes Mellitus.
    Date December 1994
    Journal The American Journal of Physiology
    Excerpt

    Because the insulin-responsive glucose transporter, GLUT4, is expressed in renal vascular and glomerular cells, we determined the effects of experimental diabetes mellitus on GLUT4 expression and glucose uptake by these tissues. Quantitative reverse-transcription polymerase chain reaction studies of microdissected afferent microvessels and renal glomeruli showed that, after 1 wk of diabetes, GLUT4 mRNA was decreased to 26 and 34% of control values, respectively. GLUT4 immunoblots of renal glomerular and microvessel samples showed that GLUT4 polypeptide was decreased to 51% of control values. These results were confirmed by indirect immunofluorescence, which showed decreased GLUT4 expression in glomerular cells and in vascular smooth muscle cells of the afferent microvasculature of diabetic animals. Uptake of the glucose analogue, 2-deoxyglucose, was also depressed in microvessels of diabetic rats to 57% of control values, supporting the conclusion that fewer total glucose transporters were available for glucose uptake into diabetic renal glomerular and microvascular cells. Thus both GLUT4 expression and glucose uptake by glomerular and microvascular cells are decreased in diabetic animals. These results have led us to suggest a mechanism by which decreased renal GLUT4 expression could contribute to glomerular hyperfiltration and hypertension seen in early diabetes.

    Title Ischemia Induces Translocation of the Insulin-responsive Glucose Transporter Glut4 to the Plasma Membrane of Cardiac Myocytes.
    Date March 1994
    Journal Circulation
    Excerpt

    Acute myocardial ischemia is accompanied by an increase in glucose uptake and metabolism, which appears to be important in protecting myocardial cells from irreversible ischemic injury. Because insulin augments myocardial glucose uptake by inducing the translocation of glucose transporters from an intracellular compartment to the plasma membrane, we hypothesized that acute ischemia would trigger a similar translocation.

    Title Cyclic Amp Selectively Increases Renin Mrna Stability in Cultured Juxtaglomerular Granular Cells.
    Date December 1993
    Journal The Journal of Biological Chemistry
    Excerpt

    This study was undertaken to examine the regulation of renin release and gene expression in primary cultures of juxtaglomerular granular (JGG) cells. JGG cells, isolated from mouse kidney, demonstrated high purity and showed regulated renin release in vitro. Changes in steady-state renin mRNA levels were assessed by quantitative polymerase chain reaction techniques, with polymerase chain reaction amplification efficiency monitored by co-amplification of experimental samples with a dilution series of cDNA for a mutant template. When the cells were incubated in the presence or absence of forskolin, isoproterenol, or 8-bromo-cAMP plus 3-isobutyl-1-methylxanthine for 24 h or cholera toxin for 12 h, renin mRNA levels were increased 3.9-, 4.4-, 5.1-, and 3.3-fold, respectively (all, p < 0.05). A significant increase in renin mRNA levels was observed 8 h after treatment with forskolin, but no change was detectable at 4 h. Cycloheximide did not prevent the increase in renin mRNA by isoproterenol. When RNA synthesis was inhibited by incubation with actinomycin D (5 micrograms/ml), renin mRNA levels declined with a half-life of 3.0 +/- 0.8 h. Treatment with forskolin increased renin mRNA half-life to 10.8 +/- 2.7 h (p < 0.025). The half-life of beta-actin, endothelin-1, or the facilitative glucose transporter-1 (GLUT-1) mRNA expressed in the same cells was not altered, although the steady-state levels of GLUT-1 mRNA increased 2.2-fold after treatment with forskolin. These data demonstrate that cAMP increases renin release and mRNA levels in JGG cells in vitro, that the stimulatory effect of cAMP on renin mRNA is delayed but does not require new protein synthesis, and that the increased renin mRNA levels induced by cAMP are due in part to a selective increase in renin mRNA stability.

    Title Endothelin-1 Mrna in Glomerular and Epithelial Cells of Kidney.
    Date November 1993
    Journal The American Journal of Physiology
    Excerpt

    To examine the question of the tubular localization of renal endothelin-1 (ET-1) mRNA, cDNA generated by reverse transcription of isolated rat tubule RNA was amplified by polymerase chain reaction using rat ET-1-specific oligonucleotides. Product identity was determined by restriction enzyme digestion or direct product sequencing. ET-1 mRNA was found to increase in renal tissue in a corticomedullary direction. High levels of ET-1 mRNA were found in dissected glomeruli and in juxtaglomerular cells in short-term primary culture. Among tubule segments, ET-1 mRNA was most abundant in inner medullary collecting ducts (IMCD), but products were also found with cDNA derived from proximal convoluted and straight tubules, thick ascending limbs, and outer medullary collecting ducts. In kidneys of untreated, homozygous Brattleboro rats, the increase of ET-1 mRNA along the corticomedullary axis as well as the preponderance of tubular ET-1 mRNA in IMCD was not observed. Our data show that ET-1 mRNA is present in all nephron segments studied and that its expression may be dependent on the functional state of the kidney. Our results are consistent with the proposal that ET-1 modifies tubular function in an autocrine or paracrine fashion.

    Title Direct Sequencing of Double-stranded Pcr Products Isolated from Conventional Agarose Gels.
    Date November 1993
    Journal Biotechniques
    Title Pharmacologic Management of Adult Idiopathic Nephrotic Syndrome.
    Date October 1993
    Journal Clinical Pharmacy
    Excerpt

    The pathophysiology, clinical features, complications, and pharmacologic management of adult idiopathic nephrotic syndrome are reviewed. Loss of plasma proteins in the urine is the primary process leading to the nephrotic syndrome, which is characterized by hypoalbuminemia, hyperlipidemia, and edema. The four principal causes, or subclasses, of adult idiopathic nephrotic syndrome are membranous nephropathy (MN), minimal change disease (MCD), focal segmental glomerulosclerosis (FSGS), and membranoproliferative glomerulonephritis (MPGN); definitive diagnosis requires histologic examination of a renal biopsy specimen. Treatment of nephrotic syndrome may be directed at the specific cause of the proteinuria, the proteinuria itself, or the complications induced by the syndrome. The four subclasses of nephrotic syndrome vary in their response to therapy. Corticosteroids, alone or in combination with cytotoxic agents, and cyclosporine have been used to induce partial or complete remission in patients with MN, MCD, and FSGS; combinations of corticosteroids, cytotoxic agents, platelet inhibitors, and anticoagulants have been used to treat patients with MPGN. Treatment of proteinuria involves dietary protein restriction with the possible addition of an angiotensin-converting-enzyme inhibitor or a nonsteroidal anti-inflammatory drug. Management of the complications of nephrotic syndrome encompasses the use of diuretics; a low-cholesterol, low-fat diet; lipid-lowering agents; and anticoagulants. Patients with nephrotic syndrome are in a constant state of flux with respect to fluid status, organ function, and critical protein balance. Treatment is based on the histologic subclass of the disease.

    Title Control of Hemorrhage During Renal Failure with Triglycyl-lysine-vasopressin.
    Date September 1993
    Journal Annals of Plastic Surgery
    Excerpt

    A 35-year-old man with chronic renal failure developed toxic epidermal necrolysis due to combination antibiotic therapy for a community acquired pneumonia. During wound care for his toxic epidermal necrolysis, he developed massive bleeding, a 4 to 6 unit blood loss at each dressing change, due to uremia-associated platelet dysfunction and thrombocytopenia. After failure of standard therapy, the man was treated with intravenous triglycyl-lysine-vasopressin, a selective peripheral vasoconstrictor. Transfusion requirements stopped during treatment. This man went on to full recovery with complete wound healing. Triglycyl-lysine-vasopressin effectively reduced skin blood loss in this man with toxic epidermal necrolysis and an intrinsic hemostatic defect, and may be useful in other patients with cutaneous blood loss.

    Title Molecular and Cellular Aspects of Diabetes Mellitus: Applications to Diabetic Nephropathy.
    Date February 1993
    Journal Seminars in Nephrology
    Title Insulin-responsive Glucose Transporter Expression in Renal Microvessels and Glomeruli.
    Date January 1993
    Journal Kidney International
    Excerpt

    The insulin-responsive glucose transporter (GLUT4) is expressed at high levels in fat and skeletal muscle, which account for the majority of insulin-stimulated glucose uptake. However, GLUT4 is also expressed at lower levels in kidney and several other tissues. We have used a variety of protein and mRNA detection techniques to determine the sites of renal GLUT4 expression. Indirect immunofluorescence experiments with two specific anti-peptide antisera detected GLUT4 in the smooth muscle cells of the rat renal microvasculature, in renal glomerulus, and in cultured glomerular mesangial and epithelial cells. PCR amplification of cDNA derived from microdissected renal glomeruli, microvessels and tubules corroborated this distribution of GLUT4, and Northern blotting demonstrated GLUT4 mRNA in cultured glomerular mesangial cells. Both the immunofluorescence and PCR data suggested that GLUT4 is most highly expressed in renal microvessels. Our results show that certain renal cells, such as renal microvascular smooth muscle cells, express the insulin-responsive glucose transporter and therefore may demonstrate altered glucose uptake and metabolism in diabetes mellitus.

    Title Intracellular Atp Can Regulate Afferent Arteriolar Tone Via Atp-sensitive K+ Channels in the Rabbit.
    Date October 1992
    Journal The Journal of Clinical Investigation
    Excerpt

    Studies were performed to assess whether ATP-sensitive K+ (KATP) channels on rabbit preglomerular vessels can influence afferent arteriolar (AA) tone. K+ channels with a slope conductance of 258 +/- 13 (n = 7) pS and pronounced voltage dependence were demonstrated in excised patches from vascular smooth muscle cells of microdissected preglomerular segments. Channel activity was markedly reduced by 1 mM ATP and in a dose-dependent fashion by glibenclamide (10(-9) M to 10(-6) M), a specific antagonist of KATP channels. 10(-5) M diazoxide, a K+ channel opener, activated these channels in the presence of ATP, and this effect was also blocked by glibenclamide. To determine the role of these KATP channels in the control of vascular tone, diazoxide was tested on isolated perfused AA. After preconstriction from a control diameter of 13.1 +/- 1.1 to 3.5 +/- 2.1 microns with phenylephrine (PE), addition of 10(-5) M diazoxide dilated vessels to 11.2 +/- 0.7 microns, which was not different from control. Further addition of 10(-5) M glibenclamide reconstricted the vessels to 5.8 +/- 1.5 microns (n = 5; P less than 0.03). In support of its specificity for KATP channels, glibenclamide did not reverse verapamil induced dilation in a separate series of experiments. To determine whether intracellular ATP levels can effect AA tone, studies were conducted to test the effect of the glycolytic inhibitor 2-deoxy-D-glucose. After preconstriction from 13.4 +/- 3.2 to 7.7 +/- 1.3 microns with PE, bath glucose was replaced with 6 mM 2-deoxy-D-glucose. Within 10 min, the arteriole dilated to a mean value of 11.8 +/- 1.4 microns (n = 6; NS compared to control). Subsequent addition of 10(-5) M glibenclamide significantly reconstricted the vessels to a diameter of 8.6 +/- 0.5 micron (P less than 0.04). These data demonstrate that KATP channels are present on the preglomerular vasculature and that changes in intracellular ATP can directly influence afferent arteriolar tone via these channels.

    Title Sulfadiazine Crystalluria Revisited. The Treatment of Toxoplasma Encephalitis in Patients with Acquired Immunodeficiency Syndrome.
    Date December 1990
    Journal Archives of Internal Medicine
    Excerpt

    Toxoplasma gondii encephalitis is an important opportunistic infection in the acquired immunodeficiency syndrome, estimated to occur in 20,000 to 40,000 patients with acquired immunodeficiency syndrome in the United States by 1991. The combination of sulfadiazine and pyrimethamine is regarded as the treatment of choice. Acute renal failure due to crystal deposition in the urinary tract was well described 30 to 40 years ago and is likely to resurface as a clinical entity if appropriate prophylactic measures are not taken. We describe two cases of sulfadiazine-induced crystalluria and renal failure in patients with acquired immunodeficiency syndrome, review the pertinent literature, and discuss the pathogenesis. Recommendations are made for the prophylaxis and treatment of sulfadiazine-related renal toxic reaction. Physicians using this "new" drug must be aware of the potential danger of sulfonamide-induced injury to the urinary tract.

    Title The Major Kidney Band 3 Gene Transcript Predicts an Amino-terminal Truncated Band 3 Polypeptide.
    Date June 1989
    Journal The Journal of Biological Chemistry
    Excerpt

    We have characterized multiple transcripts from the band 3 gene expressed in rat and mouse kidney. In each species, the major transcript lacks sequence from the first three exons of the band 3 gene. The murine transcript predicts a kidney band 3 polypeptide with a truncated amino terminus, lacking the first 79 amino acids of erythroid band 3. When expressed in Xenopus oocytes this truncated band 3 functions in anion transport.

    Title A Glucose Transport Protein Expressed Predominately in Insulin-responsive Tissues.
    Date May 1989
    Journal Proceedings of the National Academy of Sciences of the United States of America
    Excerpt

    Using low-stringency hybridization to the rat brain glucose transporter (GT), a 2489-base-pair cDNA clone was isolated from a rat soleus lambda gt10 cDNA library. It encodes a 509-amino acid protein whose sequence and predicted membrane structure is very similar to those of the rat brain and liver GTs. The muscle GT-like protein is 65% identical in amino acid sequence to the rat brain GT and 52% identical to the rat liver GT; the major differences are in the NH2- and COOH-terminal hydrophilic segments. This GT-like mRNA is expressed predominately in tissues where glucose transport is sensitive to insulin, including striated muscle, cardiac muscle, and adipose tissue; low-level expression is also detected in smooth muscle and kidney mRNA. This GT-like cDNA is the fourth member of the mammalian GT-related gene family identified to date. We propose that it encodes an insulin-sensitive GT.

    Title Bordetella Bronchiseptica Pneumonia in a Patient with Chronic Lymphocytic Leukemia.
    Date October 1987
    Journal Southern Medical Journal
    Excerpt

    This case report describes two episodes of pneumonia caused by Bordetella bronchiseptica in a patient with chronic lymphocytic leukemia. There was discrepancy between the in vitro sensitivity testing of the organism and subsequent clinical response to several antimicrobial agents. Human infection with B bronchiseptica is almost always associated with severe underlying disease and contact with an appropriate animal reservoir.

    Title Low Fractional Excretion of Sodium in Acute Renal Failure: Role of Timing of the Test and Ischemia.
    Date May 1987
    Journal American Journal of Nephrology
    Excerpt

    To evaluate the mechanisms for a low fractional excretion of Na (FENa less than or equal to 1.0) in acute renal failure (ARF) of a sustained nature, causes were determined independent of FENa in 41 patients without volume depletion, obstruction, vasculitis or glomerulonephritis. The 16 patients (39%) with low FENa had lower incidence of preexisting azotemia, lower peak serum creatinine, but higher incidence of renal ischemia and earlier testing (by 1.7 days). Seven of ten such patients converted to high FENa on repeat, whereas FENa remained high in 15 of 17 patients with initially high values. The initial FENa was a direct function of time from the onset of ARF. Low FENa in acute but sustained renal failure is therefore best explained by milder insults; earlier determinations, and/or super-imposed renal ischemia.

    Title Left Atrial-to-right Atrial Shunt Without Atrial Septal Defect or Precordial Murmur. Pulmonary Varix and Hypertrophic Cardiomyopathy.
    Date March 1982
    Journal Chest
    Title Comparison of Degree and Extent of Coronary Narrowing by Atherosclerotic Plaque in Anterior and Posterior Transmural Acute Myocardial Infarction.
    Date November 1981
    Journal Circulation
    Title Significance of Coronary Arterial Thrombus in Transmural Acute Myocardial Infarction. A Study of 54 Necropsy Patients.
    Date May 1981
    Journal Circulation
    Excerpt

    In 54 necropsy patients with transmural acute myocardial infarction (AMI) and coronary arterial thrombi, histologic sections of coronary arteries that contained the thrombi were examined by video-planimetry to determine if the amount of luminal narrowing caused by thrombi was comparable to that produced by underlying atherosclerotic plaques, and to determine the amount of luminal narrowing by plaques immediately proximal and distal to the thrombi. The 54 coronary arteries in the 54 patients were narrowed 33-98% (mean 81%) by atherosclerotic plaque alone in cross-sectional area at the site of the thrombus (occlusive in 47 and nonocclusive in seven), from 26-98% (mean 75%) within the 2-cm segment proximal to the thrombus, and from 43-98% (mean 79%) within the 2-cm segment distal to the thrombus. Of the 54 arteries, 52 (96%) were narrowed 76-98% in cross-sectional area by atherosclerotic plaque alone at or immediately proximal or distal to the thrombus and 26 (48%) were narrowed 91-98% by plaque alone. The thrombi were 0.1--6.0 mm2 (mean 1.4 mm2) in cross-sectional area and the underlying atherosclerotic plaques were 3.0-21.0 mm2 (mean 8.7 mm2). Thus, among necropsy patients with transmural AMI, coronary thrombi occur at sites already severely narrowed by atherosclerotic plaques.

    Title Radiation Heart Disease. Analysis of 16 Young (aged 15 to 33 Years) Necropsy Patients Who Received over 3,500 Rads to the Heart.
    Date May 1981
    Journal The American Journal of Medicine
    Excerpt

    Certain clinical and necropsy findings are described in 16 young (aged 15 to 33 years) patients who received greater than 3,500 rads to the heart five to 144 months before death. All 16 had some radiation-induced damage to the heart: 15 had thickened pericardia (five of whom had evidence of cardiac tamponade); eight had increased interstitial myocardial fibrosis, particularly in the right ventricle; 12 had fibrous thickening of the mural endocardium and 13 of the valvular endocardium. Except for valvular thickening, the changes were more frequent in the right side of the heart than in the left, presumably because of higher radiation doses to the anterior surface of the heart. In six of the 16 study patients and in one of 10 control subjects, one or more major epicardial coronary arteries were narrowed from 76 to 100 percent in cross-sectional area by atherosclerotic plaque; one patient had a healed myocardial infarct at necropsy and one died suddenly. In 10 patients and in the 10 control subjects, the four major epicardial coronary arteries were examined quantitatively: 6 percent of the 469 five millimeter segments of coronary artery from the patients were narrowed from 76 to 100 percent (controls = 0.2 percent, p = 0.06) and 22 percent were narrowed from 51 to 75 percent (controls = 12 percent). The proximal portion of the arteries in the patients had significantly more narrowing than the distal portions. The arterial plaques in the patients were largely composed of fibrous tissue; the media were frequently replaced by fibrous tissue, and the adventitia were often densely thickened by fibrous tissue. In five patients, there was focal thickening (with or without luminal narrowing) of the intramural coronary arteries. Thus, radiation to the heart may produce a wide spectrum of functional and anatomic changes but particularly damage to the pericardia and the underlying epicardial coronary arteries.

    Title Coronary Artery Disease in the Hurler Syndrome. Qualitative and Quantitative Analysis of the Extent of Coronary Narrowing at Necropsy in Six Children.
    Date April 1981
    Journal The American Journal of Cardiology
    Excerpt

    The amount of cross-sectional area luminal narrowing in each 5 mm segment of each of the four major epicardial coronary arteries (right, left main, left anterior descending and left circumflex) is described at necropsy in six children (aged 3 to 16 years) with the Hurler syndrome. In five patients at least one of the four major coronary arteries was narrowed 76 to 100 percent, and in four of these five patients all four major arteries were narrowed to this extent. Of the 24 major coronary arteries in the six patients, 17 (71 percent) were narrowed 76 to 100 percent at some point. A total of 182 segments were examined from the 24 major coronary arteries, and the extent of narrowing was as follows: 96 to 100 percent, 14 (8 percent); 76 to 95 percent, 61 (34 percent); 51 to 75 percent, 59 (32 percent); 26 to 50 percent, 39 (21 percent) and 0 to 25 percent, 9 (5 percent). By applying a score of 1 to 4 to each 5 mm segment according to its category of narrowing (1 = 0 to 25 percent; 2 = 26 to 50 percent; 3 = 51 to 75 percent and 4 = 76 to 100 percent), the 182 segments had a total score of 570 and a mean score of 3.2, indicating that each segment was narrowed an average of about 67 percent in cross-sectional area. Thus, narrowing of the major epicardial coronary arteries at necropsy is usually diffuse and severe in the Hurler syndrome, which is the cause of the most severe coronary narrowing in childhood.

    Title Structure-function Correlations in Cardiovascular and Pulmonary Diseases (cpc). Death in the Disco.
    Date October 1980
    Journal Chest
    Title Emergency Cardiac Surgery. Ventricular Septal Defect and Ventricular Aneurysm.
    Date December 1974
    Journal The Journal of the Kansas Medical Society
    Title Abnormalities in Signaling Pathways in Diabetic Nephropathy.
    Date
    Journal Expert Review of Endocrinology & Metabolism
    Excerpt

    Diabetic nephropathy (DN) is characterized by a plethora of signaling abnormalities that together ultimately result in the clinical and pathologic hallmarks of DN, namely progressive albuminuria followed by a gradual decline in glomerular filtration rate leading to kidney failure, and accompanied by podocyte loss, progressive glomerular sclerosis and, ultimately, progressive tubulointerstitial fibrosis. Over the past few years, the general understanding of the abnormalities in signaling pathways that lead to DN has expanded considerably. In this review, some of the important pathways that appear to be involved in driving this process are discussed, with special emphasis on newer findings and insights. Newer concepts regarding signaling changes in bradykinin, mTOR, JAK/STAT, MCP-1, VEGF, endothelial nitric oxide synthase, activated protein C and other pathways are discussed.

    Title The Glomerular Podocyte As a Target of Growth Hormone Action: Implications for the Pathogenesis of Diabetic Nephropathy.
    Date
    Journal Current Diabetes Reviews
    Excerpt

    Involvement of the growth hormone (GH) / insulin-like growth factor 1 (IGF-I) axis in the pathogenesis of diabetic nephropathy (DN) is strongly suggested by studies investigating the impact of GH excess and deficiency on renal structure and function. GH excess in both the human (acromegaly) and in transgenic animal models is characterized by significant structural and functional changes in the kidney. In the human a direct relationship has been noted between the activity of the GH/IGF-1 axis and renal hypertrophy, microalbuminuria, and glomerulosclerosis. Conversely, states of GH deficiency or deficiency or inhibition of GH receptor (GHR) activity confer a protective effect against DN. The glomerular podocyte plays a central and critical role in the structural and functional integrity of the glomerular filtration barrier and maintenance of normal renal function. Recent studies have revealed that the glomerular podocyte is a target of GH action and that GH's actions on the podocyte could be detrimental to the structure and function of the podocyte. These results provide a novel mechanism for GH's role in the pathogenesis of DN and offer the possibility of targeting the GH/IGF-1 axis for the prevention and treatment of DN.

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