Matrix metalloproteinase-9 regulates afferent arteriolar remodeling and function in hypertension-induced kidney disease.

This study tested if matrix metalloproteinase (MMP)-9 promoted microvascular pathology that initiates hypertensive (HT) kidney disease in salt-sensitive (SS) Dahl rats. SS rats lacking Mmp9 (Mmp9-/-) and littermate control SS rats were studied after one week on a normotensive 0.3% sodium chloride (Pre-HT SS and Pre-HT Mmp9-/-) or a hypertension-inducing diet containing 4.0% sodium chloride (HT SS and HT Mmp9-/-). Telemetry-monitored blood pressure of both the HT SS and HT Mmp9-/- rats increased and did not differ. Kidney microvessel transforming growth factor-beta 1 (Tgfb1) mRNA did not differ between Pre-HT SS and Pre-HT Mmp9-/- rats, but with hypertension and expression of Mmp9 and Tgfb1 increased in HT SS rats, along with phospho-Smad2 labeling of nuclei of vascular smooth muscle cells, and with peri-arteriolar fibronectin deposition. Loss of MMP-9 prevented hypertension-induced phenotypic transformation of microvascular smooth muscle cells and the expected increased microvascular expression of pro-inflammatory molecules. Loss of MMP-9 in vascular smooth muscle cells in vitro prevented cyclic strain-induced production of active TGF-ß1 and phospho-Smad2/3 stimulation. Afferent arteriolar autoregulation was impaired in HT SS rats but not in HT Mmp9-/- rats or the HT SS rats treated with doxycycline, an MMP inhibitor. HT SS but not HT Mmp9-/- rats showed decreased glomerular Wilms Tumor 1 protein-positive cells (a marker of podocytes) along with increased urinary podocin and nephrin mRNA excretion, all indicative of glomerular damage. Thus, our findings support an active role for MMP-9 in a hypertension-induced kidney microvascular remodeling process that promotes glomerular epithelial cell injury in SS rats.

Endothelial cells overexpressing CXCR1/2 are renoprotective in rats with acute kidney injury.

Inflammation that develops with the release of chemokines and cytokines during acute kidney injury (AKI) has been shown to participate in functional renal recovery. Although a major research focus has been on the role of macrophages, the family of C-X-C motif chemokines that promote neutrophil adherence and activation also increases with kidney ischemia-reperfusion (I/R) injury. This study tested the hypothesis that intravenous delivery of endothelial cells (ECs) that overexpress (C-X-C motif) chemokine receptors 1 and 2 (CXCR1 and CXCR2, respectively) improves outcomes in kidney I/R injury. Overexpression of CXCR1/2 enhanced homing of endothelial cells to I/R-injured kidneys and limited interstitial fibrosis, capillary rarefaction, and tissue injury biomarkers (serum creatinine concentration and urinary kidney injury molecule-1) following AKI and also reduced expression of P-selectin and the rodent (C-X-C motif) chemokine cytokine-induced neutrophil chemoattractant (CINC)-2ß as well as the number of myeloperoxidase-positive cells in the postischemic kidney. The serum chemokine/cytokine profile, including CINC-1, showed similar reductions. These findings were not observed in rats given endothelial cells transduced with an empty adenoviral vector (null-ECs) or a vehicle alone. These data indicate that extrarenal endothelial cells that overexpress CXCR1 and CXCR2, but not null-ECs or vehicle alone, reduce I/R kidney injury and preserve kidney function in a rat model of AKI.NEW & NOTEWORTHY Inflammation facilitates kidney ischemia-reperfusion (I/R) injury. Endothelial cells (ECs) that were modified to overexpress (C-X-C motif) chemokine receptor (CXCR)1/2 (CXCR1/2-ECs) were injected immediately following kidney I/R injury. The interaction of CXCR1/2-ECs, but not ECs transduced with an empty adenoviral vector, with injured kidney tissue preserved kidney function and reduced production of inflammatory markers, capillary rarefaction, and interstitial fibrosis. The study highlights a functional role for the C-X-C chemokine pathway in kidney damage following I/R injury.

Effect of Hysteroscopic Polypectomy Combined with Mirena Placement on Postoperative Adverse Reactions and Recurrence Rate of Endometrial Polyps: Based on a Large-Sample, Single-Center, Retrospective Cohort Study.

Objective: To investigate the effect of hysteroscopy surgery combined with Mirena on postoperative adverse reactions and recurrence rate of endometrial polyps (EP). Methods: A total of 312 patients who underwent hysteroscopic polypectomy of EP in our hospital from June 2017 to November 2020 were enrolled retrospectively. Among them, 42 patients did not take any treatment after the operation (control group), 156 patients were treated with levonorgestrel intrauterine birth control system (Mirena group), and 114 patients were treated with oral spironolone ethinylestradiol tablets (oral group). The clinical data of 312 patients were recorded and followed up regularly. All patients were followed up through an outpatient clinic or telephone to 12 months after the operation. The patients' age, disease course, number of pregnancies, clinical manifestations, endometrial thickness before the operation, duration of operation, amount of bleeding during the operation, and number and size of polyps were analyzed. The recurrence and postoperative side effects of EP in the three groups were followed up within 12 months after the operation. Results: There was no significant difference in endometrial thickness among the three groups before treatment (P > 0.05). After 3 months, 6 months, and 12 months of treatment, the endometrial thickness of the three groups decreased, while the decrease in the Mirena group and the oral group was better compared to the control (P < 0.05). The decrease in the Mirena group was better than that in the oral group (P < 0.05). There was no significant difference in hemoglobin levels among the three groups before treatment (P > 0.05). After 3, 6, and 12 months of treatment, the hemoglobin levels of the three groups increased to varying degrees, while the levels of the Mirena group and oral group were better compared to the control (P < 0.05). Three months after the operation, the improvement of clinical symptoms was similar in the three groups, and there was no significant difference among the three groups (P > 0.05). At 6 and 12 months after the operation, the improvement of clinical symptoms in the oral group and Mirena group was better compared to the control group (P < 0.05), but there was no significant difference between the oral group and Mirena group (P > 0.05). After the operation, some patients had complications such as lower abdominal pain, breast distension pain, irregular vaginal bleeding, and abnormal liver function. There was no significant difference in the number of complications among the three groups (P > 0.05). During the follow-up to 12 months after the operation, the recurrence rate in the oral group and Mirena group was lower compared to the control (P < 0.05), and the recurrence rate in the Mirena group was lower than that in the oral group (P < 0.05). Conclusion: Placing Mirena immediately after hysteroscopic polypectomy of EP can reduce the recurrence rate of endometrial polyps, increase the level of hemoglobin, and reduce the thickness of the endometrium, which can be employed and popularized according to the condition of patients in clinical work.

Dietary salt initiates redox signaling between endothelium and vascular smooth muscle through NADPH oxidase 4.

Prevention of phenotype switching of vascular smooth muscle cells is an important determinant of normal vascular physiology. Hydrogen peroxide (H2O2) promotes osteogenic differentiation of vascular smooth muscle cells through expression of Runt related transcription factor 2 (Runx2). In this study, an increase in dietary NaCl increased endothelial H2O2 generation through NOX4, a NAD(P)H oxidase. The production of H2O2 was sufficient to increase Runx2, osteopontin and osteocalcin in adjacent vascular smooth muscle cells from control littermate mice but was inhibited in mice lacking endothelial Nox4. A vascular smooth muscle cell culture model confirmed the direct involvement of the activation of protein kinase B (Akt) with inactivation of FoxO1 and FoxO3a observed in the control mice on the high NaCl diet. The present study also showed a reduction of catalase activity in aortas during high NaCl intake. The findings demonstrated an interesting cell-cell communication in the vascular wall that was initiated with H2O2 production by endothelium and was regulated by dietary NaCl intake. A better understanding of how dietary salt intake alters vascular biology may improve treatment of vascular disease that involves activation of Runx2.

Cellular antioxidant mechanisms control immunoglobulin light chain-mediated proximal tubule injury.

A major cause of morbidity and mortality in multiple myeloma is kidney injury from overproduction of monoclonal immunoglobulin light chains (FLC). FLC can induce damage through the production of hydrogen peroxide, which activates pro-inflammatory and pro-apoptotic pathways. The present study focused on catalase, a highly conserved antioxidant enzyme that degrades hydrogen peroxide. Initial findings were that FLC increased hydrogen peroxide levels but also decreased catalase levels and activity in proximal tubule epithelium. In order to clarify, we showed that the phosphatidylinositol 3-kinase inhibitor, LY294002, inhibited FLC-induced Akt-mediated deactivation of Forkhead box O class 3a (FoxO3a) and increased catalase activity in proximal tubule cells. Augmented catalase activity decreased FLC-mediated production of hydrogen peroxide as well as the associated increase in High Mobility Group Box 1 (HMGB1) protein release and caspase-3 activity. Coincubation of cells with FLC and an allosteric activator of Sirtuin 1 (SIRT1) was also sufficient to increase catalase activity and promote similar cytoprotective effects. Our studies confirmed that the mechanism of downregulation of catalase by FLC involved deactivation of FoxO3a and inhibition of SIRT1. Mechanistic understanding of catalase regulation allows for future treatments that target pathways that increase catalase in the setting of proximal tubule injury from FLC.

Avian erythroblastosis virus E26 oncogene homolog-1 (ETS-1) plays a role in renal microvascular pathophysiology in the Dahl salt-sensitive rat.

Prior studies reported that haploinsufficiency of the transcription factor ETS-1 is renoprotective in Dahl salt-sensitive rats, but the mechanism is unclear. Here, we tested whether ETS-1 is involved in hypertension-induced renal microvascular pathology and autoregulatory impairment. Hypertension was induced in salt-sensitive rats and salt-sensitive rats that are heterozygous with 1 wild-type or reference allele of Ets1 (SSEts1+/-) by feeding a diet containing 4% sodium chloride for 1 week. Increases in blood pressure did not differ. However, phosphorylated ETS-1 increased in afferent arterioles of hypertensive salt-sensitive rats, but not in hypertensive SSEts1+/- rats. Afferent arterioles of hypertensive salt-sensitive rats showed increased monocyte chemotactic protein-1 expression and infiltration of CD68 positive monocytes/macrophages. Isolated kidney microvessels showed increased mRNA expression of vascular cell adhesion molecule, intercellular adhesion molecule, P-selectin, fibronectin, transforming growth factor-ß, and collagen I in hypertensive salt-sensitive rats compared with hypertensive SSEts1+/- rats. Using the in vitro blood-perfused juxtamedullary nephron preparation, pressure-mediated afferent arteriolar responses were significantly blunted in hypertensive salt-sensitive rats compared to hypertensive SSEts1+/- rats. Over a 65-170 mm Hg pressure range tested baseline arteriolar diameters averaged 15.1 µm and remained between 107% and 89% of baseline diameter in hypertensive salt-sensitive rats vs. 114% and 73% in hypertensive SSEts1+/- rats (significantly different). Thus, ETS-1 participates in renal arteriolar pathology and autoregulation and thereby is involved in hypertension-mediated kidney injury in salt-sensitive rats.

Induced pluripotent stem cell-derived endothelial cells attenuate lipopolysaccharide-induced acute lung injury.

The chemokine receptors CXCR1/2 and CCR2/5 play a critical role in neutrophil and monocyte recruitment to sites of injury and/or inflammation. Neutrophil-mediated inflammation and endothelial cell (EC) injury are unifying factors in the pathogenesis of the acute respiratory distress syndrome. This study tested the hypothesis that systemic administration of rat-induced pluripotent stem cell (iPS)-derived ECs (iPS-ECs) overexpressing CXCR1/2 or CCR2/5 attenuates lipopolysaccharide (LPS)-induced acute lung injury. Rat iPS-ECs were transduced with adenovirus containing cDNA of CXCR1/2 or CCR2/5. Ovariectomized Sprague-Dawley rats (10 wk old) received intraperitoneal injection of LPS and intravenous infusion of 1) saline vehicle, 2) AdNull-iPS-ECs (iPS-ECs transduced with empty adenoviral vector), 3) CXCR1/2-iPS-ECs (iPS-ECs overexpressing CXCR1/2), or 4) CCR2/5-iPS-ECs (iPS-ECs overexpressing CCR2/5) at 2 h post-LPS. Rats receiving intraperitoneal injection of saline served as sham controls. Later (4 h), proinflammatory cytokine/chemokine mRNA and protein levels were measured in total lung homogenates by real-time RT-PCR and Luminex multiplex assays, and neutrophil and macrophage infiltration in alveoli was measured by immunohistochemical staining. Pulmonary microvascular permeability was assessed by the Evans blue technique, and pulmonary edema was estimated by wet-to-dry lung weight ratios. Albumin levels and neutrophil counts were assessed in bronchoalveolar lavage fluid at 24 h post-LPS. Both CXCR1/2-iPS-ECs and CCR2/5-iPS-ECs significantly reduced LPS-induced proinflammatory mediator expression, neutrophil and macrophage infiltration, pulmonary edema, and vascular permeability compared with controls. These provocative findings provide strong evidence that targeted delivery of iPS-ECs overexpressing CXCR1/2 or CCR2/5 prevents LPS-induced acute lung injury.NEW & NOTEWORTHY We have developed a novel approach to address neutrophil-mediated inflammation and endothelial damage by targeted delivery of rat-induced pluripotent stem cell (iPS)-derived endothelial cell (ECs)overexpressing chemokine receptors CXCR1/2 and CCR2/5 in injured lung tissue in a model of acute lung injury. We have demonstrated that intravenously transfused CXCR1/2-iPS-ECs and CCR2/5-iPS-ECs are recruited to lipopolysaccharide-injured lungs and attenuate lipopolysaccharide-induced parenchymal lung injury responses, including inflammatory mediator expression, inflammatory cell infiltration, and vascular leakage compared with controls.

Immunoglobulin light chains generate proinflammatory and profibrotic kidney injury.

Because of the less-than-robust response to therapy and impact on choice of optimal chemotherapy and prognosis, chronic kidney disease has drawn attention in the treatment of multiple myeloma, a malignant hematologic disorder that can produce significant amounts of monoclonal immunoglobulin free light chains (FLCs). These low-molecular-weight proteins are relatively freely filtered through the glomerulus and are reabsorbed by the proximal tubule. The present study demonstrated that during the process of metabolism of immunoglobulin FLCs, ROS activated the STAT1 pathway in proximal tubule epithelium. STAT1 activation served as the seminal signaling molecule that produced the proinflammatory molecule IL-1ß, as well as the profibrotic agent TGF-ß by this portion of the nephron. These effects occurred in vivo and were produced specifically by the generation of hydrogen peroxide by the VL domain of the light chain. To the extent that the experiments reflect the human condition, these studies offer insights into the pathogenesis of progressive kidney failure in the setting of lymphoproliferative disorders, such as multiple myeloma, that feature increased circulating levels of monoclonal immunoglobulin fragments that require metabolism by the kidney.

Ferritin Light Chain Confers Protection Against Sepsis-Induced Inflammation and Organ Injury.

Despite the prevalence and recognition of its detrimental impact, clinical complications of sepsis remain a major challenge. Here, we investigated the effects of myeloid ferritin heavy chain (FtH) in regulating the pathogenic sequelae of sepsis. We demonstrate that deletion of myeloid FtH leads to protection against lipopolysaccharide-induced endotoxemia and cecal ligation and puncture (CLP)-induced model of sepsis as evidenced by reduced cytokine levels, multi-organ dysfunction and mortality. We identified that such protection is predominantly mediated by the compensatory increase in circulating ferritin (ferritin light chain; FtL) in the absence of myeloid FtH. Our in vitro and in vivo studies indicate that prior exposure to ferritin light chain restrains an otherwise dysregulated response to infection. These findings are mediated by an inhibitory action of FtL on NF-κB activation, a key signaling pathway that is implicated in the pathogenesis of sepsis. We further identified that LPS mediated activation of MAPK pathways, specifically, JNK, and ERK were also reduced with FtL pre-treatment. Taken together, our findings elucidate a crucial immunomodulatory function for circulating ferritin that challenges the traditional view of this protein as a mere marker of body iron stores. Accordingly, these findings will stimulate investigations to the adaptive nature of this protein in diverse clinical settings.

Cutaneous exposure to lewisite causes acute kidney injury by invoking DNA damage and autophagic response.

Lewisite (2-chlorovinyldichloroarsine) is an organic arsenical chemical warfare agent that was developed and weaponized during World Wars I/II. Stockpiles of lewisite still exist in many parts of the world and pose potential environmental and human health threat. Exposure to lewisite and similar chemicals causes intense cutaneous inflammatory response. However, morbidity and mortality in the exposed population is not only the result of cutaneous damage but is also a result of systemic injury. Here, we provide data delineating the pathogenesis of acute kidney injury (AKI) following cutaneous exposure to lewisite and its analog phenylarsine oxide (PAO) in a murine model. Both agents caused renal tubular injury, characterized by loss of brush border in proximal tubules and tubular cell apoptosis accompanied by increases in serum creatinine, neutrophil gelatinase-associated lipocalin, and kidney injury molecule-1. Interestingly, lewisite exposure enhanced production of reactive oxygen species (ROS) in the kidney and resulted in the activation of autophagic and DNA damage response (DDR) signaling pathways with increased expression of beclin-1, autophagy-related gene 7, and LC-3A/B-II and increased phosphorylation of γ-H2A.X and checkpoint kinase 1/2, respectively. Terminal deoxyribonucleotide-transferase-mediated dUTP nick-end labeling-positive cells were detected in renal tubules along with enhanced proapoptotic BAX/cleaved caspase-3 and reduced antiapoptotic BCL2. Scavenging ROS by cutaneous postexposure application of the antioxidant N-acetyl-l-cysteine reduced lewisite-induced autophagy and DNA damage. In summary, we provide evidence that topical exposure to lewisite causes AKI. The molecular mechanism underlying these changes involves ROS-dependent activation of autophagy and DDR pathway associated with the induction of apoptosis.

Haploinsufficiency of the Transcription Factor Ets-1 Is Renoprotective in Dahl Salt-Sensitive Rats.

Studies using Dahl salt-sensitive (SS) rats identified specific quantitative trait loci that predispose animals to hypertension-associated albuminuria and kidney injury. We explored the hypothesis that kidney-specific expression of the transcription factor Ets-1, located within one of these loci on chromosome 8, mediates glomerular injury in SS hypertension. During the first week on a high-salt diet, SS rats and SS rats with only one functioning Ets-1 gene (ES rats) demonstrated similar increases in BP. However, serum creatinine concentration, albuminuria, and glomerular expression of ETS-1 and two ETS-1 targets, MCP-1 and MMP2, did not increase as substantially in ES rats as in SS rats. Mean BP subsequently increased further in SS rats and remained higher than that of ES rats for the rest of the study. After 4 weeks of high-salt intake, ES rats still showed a lower mean serum creatinine concentration and less albuminuria, as well as less histologic evidence of glomerular injury and kidney fibrosis, than SS rats did. To investigate the specific contribution of renal Ets-1, we transplanted kidneys from ES or SS rats into salt-resistant SS-Chr 13BN/McwiCrl (SS-13BN) rats. Within 10 days on a high-salt diet, BP increased similarly in ES and SS allograft recipients, becoming significantly higher than the BP of control isograft recipients. However, mean serum creatinine concentration and albuminuria remained lower in ES allograft recipients than in SS allograft recipients at 2 weeks, and ES allografts showed less glomerular injury and interstitial fibrosis. In conclusion, reduced renal expression of ETS-1 prevented hypertension-associated kidney injury in SS rats.

Transcription factor avian erythroblastosis virus E26 oncogen homolog-1 is a novel mediator of renal injury in salt-sensitive hypertension.

Transcription factor E26 transformation-specific sequence-1 (ETS-1) is a transcription factor that regulates the expression of a variety of genes, including growth factors, chemokines, and adhesion molecules. We recently demonstrated that angiotensin II increases the glomerular expression of ETS-1 and that blockade of ETS-1 ameliorates the profibrotic and proinflammatory effects of angiotensin II. The Dahl salt-sensitive rat is a paradigm of salt-sensitive hypertension associated with local activation of the renin-angiotensin system. In these studies, we determined whether: (1) salt-sensitive hypertension is associated with renal expression of ETS-1 and (2) ETS-1 participates in the development of end-organ injury in salt-sensitive hypertension. Dahl salt-sensitive rats were fed a normal-salt diet (0.5% NaCl diet) or a high-salt diet (4% NaCl) for 4 weeks. Separate groups on high-salt diet received an ETS-1 dominant-negative peptide (10 mg/kg/d), an inactive ETS-1 mutant peptide (10 mg/kg/d), the angiotensin II type 1 receptor blocker candesartan (10 mg/kg/d), or the combination high-salt diet/dominant-negative peptide/angiotensin II type 1 receptor blocker for 4 weeks. High-salt diet rats had a significant increase in the glomerular expression of the phosphorylated ETS-1 that was prevented by angiotensin II type 1 receptor blocker. ETS-1 blockade reduced proteinuria, glomerular injury score, fibronectin expression, urinary transforming growth factor-ß excretion, and macrophage infiltration. Angiotensin II type 1 receptor blocker reduced proteinuria, glomerular injury score, and macrophage infiltration, whereas concomitant ETS-1 blockade and angiotensin II type 1 receptor blocker had additive effects and reduced interstitial fibrosis. Our studies demonstrated that salt-sensitive hypertension results in increased glomerular expression of phosphorylated ETS-1 and suggested that ETS-1 plays an important role in the pathogenesis of end-organ injury in salt-sensitive hypertension.

The transcription factor E26 transformation-specific sequence-1 mediates neointima formation in arteriovenous fistula.

Hemodialysis vascular access dysfunction contributes to increased morbidity and mortality in hemodialysis patients. Arteriovenous fistula (AVF) is the preferred type of vascular access for hemodialysis but has high rates of dysfunction, in part because of excessive neointima formation. The transcription factor E26 transformation-specific sequence-1 (ETS-1) is a mediator of proinflammatory responses in hypertension and endovascular injury. We examined the role of ETS-1 in the formation of neointima in AVF. Right carotid artery to internal jugular vein fistulas were created in C57BL/6 mice and assigned to treatment with an ETS-1-dominant negative peptide (ETS-DN), an inactive mutant peptide (ETS-MU), or vehicle (n=6 per group). After 7 and 21 days, AVFs or contralateral internal jugular veins were processed for PCR, immunofluorescence, immunohistochemistry, and morphometry. In AVFs, ETS-1 mRNA increased 2.5-fold at 7 days and 4-fold at 21 days. By immunofluorescence, we confirmed increased expression of ETS-1 predominantly in the neointima and overlying endothelium. Similarly, ETS-1 expression increased in human AVFs compared with normal veins. In mice, ETS-DN, but not ETS-MU, reduced neointima formation at days 7 and 21 and reduced the expression of nitric oxide synthase 2, NADPH oxidase (NOX) 2, NOX4, E-selectin, and monocyte chemotactic protein-1. Shear stress increased ETS-1 phosphorylation in human umbilical vein cells in a NOX-dependent manner, demonstrating a role for reactive oxygen species in ETS-1 activation. These results unveil the role of ETS-1 as a mediator of neointima formation in AVF and may result in the development of novel strategies for the treatment of AVF dysfunction.

Role of the transcription factor erythroblastosis virus E26 oncogen homolog-1 (ETS-1) as mediator of the renal proinflammatory and profibrotic effects of angiotensin II.

Angiotensin II (Ang II) plays a major role in the pathogenesis of end-organ injury in hypertension via its diverse hemodynamic and nonhemodynamic effects. Erythroblastosis virus E26 oncogen homolog-1 (ETS-1) is an important transcription factor recently recognized as an important mediator of cell proliferation, inflammation, and fibrosis. In the present studies, we tested the hypothesis that ETS-1 is a common mediator of the renal proinflammatory and profibrotic effects of Ang II. C57BL6 mice (n=6 per group) were infused with vehicle (control), Ang II (1.4 mg/kg per day), Ang II and an ETS-1 dominant-negative peptide (10 mg/kg per day), or Ang II and an ETS-1 mutant peptide (10 mg/kg per day) via osmotic minipump for 2 or 4 weeks. The infusion of Ang II resulted in significant increases in blood pressure and left ventricular hypertrophy, which were not modified by ETS-1 blockade. The administration of ETS-1 dominant-negative peptide significantly attenuated Ang II-induced renal injury as assessed by urinary protein excretion, mesangial matrix expansion, and cell proliferation. Furthermore, ETS-1 dominant-negative peptide but not ETS-1 mutant peptide significantly reduced Ang II-mediated upregulation of transforming growth factor-ß, connective tissue growth factor, and α-smooth muscle actin. In addition, ETS-1 blockade reduced several proinflammatory effects of Ang II, including macrophage infiltration, nitrotyrosine expression, and NOX4 mRNA expression. Our studies suggest that ETS-1 is a common mediator of the proinflammatory and profibrotic effects of Ang II-induced hypertensive renal damage and may result in the development of novel strategies in the treatment and prevention of end-organ injury in hypertension.

Estrogen modulates NFκB signaling by enhancing IκBα levels and blocking p65 binding at the promoters of inflammatory genes via estrogen receptor-ß.

BACKGROUND: NFκB signaling is critical for expression of genes involved in the vascular injury response. We have shown that estrogen (17ß-estradiol, E2) inhibits expression of these genes in an estrogen receptor (ER)-dependent manner in injured rat carotid arteries and in tumor necrosis factor (TNF)-α treated rat aortic smooth muscle cells (RASMCs). This study tested whether E2 inhibits NFκB signaling in RASMCs and defined the mechanisms. METHODOLOGY/PRINCIPAL FINDINGS: TNF-α treated RASMCs demonstrated rapid degradation of IκBα (10-30 min), followed by dramatic increases in IκBα mRNA and protein synthesis (40-60 min). E2 enhanced TNF-α induced IκBα synthesis without affecting IκBα degradation. Chromatin immunoprecipitation (ChIP) assays revealed that E2 pretreatment both enhanced TNF-α induced binding of NFκB p65 to the IκBα promoter and suppressed TNF-α induced binding of NFκB p65 to and reduced the levels of acetylated histone 3 at promoters of monocyte chemotactic protein (MCP)-1 and cytokine-induced neutrophil chemoattractant (CINC)-2ß genes. ChIP analyses also demonstrated that ERß can be recruited to the promoters of MCP-1 and CINC-2ß during co-treatment with TNF-α and E2. CONCLUSIONS: These data demonstrate that E2 inhibits inflammation in RASMCs by two distinct mechanisms: promoting new synthesis of IκBα, thus accelerating a negative feedback loop in NFκB signaling, and directly inhibiting binding of NFκB to the promoters of inflammatory genes. This first demonstration of multifaceted modulation of NFκB signaling by E2 may represent a novel mechanism by which E2 protects the vasculature against inflammatory injury.

Cardiomyopathy and response to enzyme replacement therapy in a male mouse model for Fabry disease.

Fabry disease is an X-linked disorder of glycosphingolipid metabolism that results in progressive accumulation of neutral glycosphingolipids, (predominately globotriaosylceramide; GL-3) in lysosomes, as well as other cellular compartments and the extracellular space. Our aim was to characterize the cardiac phenotype of male knock-out mice that are deficient in alpha-galactosidase A activity, as a model for Fabry disease and test the efficacy of Enzyme Replacement Therapy with agalsidase-beta. Male mice (3-4 months of age) were characterized with awake blood pressure and heart rate measurements, cardiac echocardiography and electrocardiography measurements under light anesthesia, histological studies and molecular studies with real-time polymerase chain reaction. The Fabry knock-out mouse has bradycardia and lower blood pressure than control wild type (CB7BL/6J) mice. In Fabry knock-out mice, the cardiomyopathy associated mild hypertrophy at echography with normal systolic LV function and mild diastolic dysfunction. Premature atrial contractions were more frequent in without conduction defect. Heart weight normalized to tibial length was increased in Fabry knock-out mice. Ascending aorta dilatation was observed. Molecular studies were consistent with early stages of cardiac remodeling. A single dose of agalsidase-beta (3 mg/kg) did not affect the LV hypertrophy, function or heart rate, but did improve the mRNA signals of early cardiac remodeling. In conclusion, the alpha-galactosidase A deficient mice at 3 to 4 months of age have cardiac and vascular alterations similar to that described in early clinical stage of Fabry disease in children and adolescents. Enzyme replacement therapy affects cardiac molecular remodeling after a single dose.

Nicotine exposure and the progression of chronic kidney disease: role of the α7-nicotinic acetylcholine receptor.

Clinical studies have established the role of cigarette smoking as a risk factor in the progression of chronic kidney disease (CKD). We have shown that nicotine promotes mesangial cell proliferation and hypertrophy via nonneuronal nicotinic acetylcholine receptors (nAChRs). The α7-nAChR is one of the most important subunits of the nAChRs. These studies were designed to test the hypothesis that nicotine worsens renal injury in rats with 5/6 nephrectomy (5/6Nx) and that the α7-nAChR subunit is required for these effects. We studied five different groups: Sham, 5/6Nx, 5/6Nx + nicotine (Nic; 100 µg/ml dry wt), 5/6Nx + Nic + α7-nAChR blocker methyllicaconitine (MLA; 3 mg·kg(-1)·day(-1) sq), and Sham + Nic. Blood pressure was measured by the tail-cuff method, and urine was collected for proteinuria. After 12 wk, the rats were euthanized and kidneys were collected. We observed expression of the α7-nAChR in the proximal and distal tubules. The administration of nicotine induced a small increase in blood pressure and resulted in cotinine levels similar to those found in the plasma of smokers. In 5/6Nx rats, the administration of nicotine significantly increased urinary protein excretion (onefold), worsened the glomerular injury score and increased fibronectin (∼ 50%), NADPH oxidase 4 (NOX4; ∼100%), and transforming growth factor-ß expression (∼200%). The administration of nicotine to sham rats increased total proteinuria but not albuminuria, suggesting direct effects on tubular protein reabsorption. These effects were prevented by MLA, demonstrating a critical role for the α7-nAChR as a mediator of the effects of nicotine in the progression of CKD.

The transcription factor ETS-1 regulates angiotensin II-stimulated fibronectin production in mesangial cells.

Angiotensin II (ANG II) produced as result of activation of the renin-angiotensin system (RAS) plays a critical role in the pathogenesis of chronic kidney disease via its hemodynamic effects on the renal microcirculation as well as by its nonhemodynamic actions including the production of extracellular matrix proteins such as fibronectin, a multifunctional extracellular matrix protein that plays a major role in cell adhesion and migration as well as in the development of glomerulosclerosis. ETS-1 is an important transcription factor essential for normal kidney development and glomerular integrity. We previously showed that ANG II increases ETS-1 expression and is required for fibronectin production in mesangial cells. In these studies, we determined that ANG II induces phosphorylation of ETS-1 via activation of the type 1 ANG II receptor and that Erk1/2 and Akt/PKB phosphorylation are required for these effects. In addition, we characterized the role of ETS-1 on the transcriptional activation of fibronectin production in mesangial cells. We determined that ETS-1 directly activates the fibronectin promoter and by utilizing gel shift assays and chromatin immunoprecipitation assays identified two different ETS-1 binding sites that promote the transcriptional activation of fibronectin in response to ANG II. In addition, we identified the essential role of CREB and its coactivator p300 on the transcriptional activation of fibronectin by ETS-1. These studies unveil novel mechanisms involved in RAS-induced production of the extracellular matrix protein fibronectin in mesangial cells and establish the role of the transcription factor ETS-1 as a direct mediator of these effects.

O-GlcNAc modification of NFκB p65 inhibits TNF-α-induced inflammatory mediator expression in rat aortic smooth muscle cells.

BACKGROUND: We have shown that glucosamine (GlcN) or O-(2-acetamido-2-deoxy-D-glucopyranosylidene)amino-N-phenylcarbamate (PUGNAc) treatment augments O-linked-N-acetylglucosamine (O-GlcNAc) protein modification and attenuates inflammatory mediator expression, leukocyte infiltration and neointima formation in balloon injured rat carotid arteries and have identified the arterial smooth muscle cell (SMC) as the target cell in the injury response. NFκB signaling has been shown to mediate the expression of inflammatory genes and neointima formation in injured arteries. Phosphorylation of the p65 subunit of NFκB is required for the transcriptional activation of NFκB. This study tested the hypothesis that GlcN or PUGNAc treatment protects vascular SMCs against tumor necrosis factor (TNF)-α induced inflammatory stress by enhancing O-GlcNAcylation and inhibiting TNF-α induced phosphorylation of NFκB p65, thus inhibiting NFκB signaling. METHODOLOGY/PRINCIPAL FINDINGS: Quiescent rat aortic SMCs were pretreated with GlcN (5 mM), PUGNAc (10(-4) M) or vehicle and then stimulated with TNF-α (10 ng/ml). Both treatments inhibited TNF-α-induced expression of chemokines [cytokine-induced neutrophil chemoattractant (CINC)-2ß and monocyte chemotactic protein (MCP)-1] and adhesion molecules [vascular cell adhesion molecule (VCAM)-1 and P-Selectin]. Both treatments inhibited TNF-α induced NFκB p65 activation and promoter activity, increased NFκB p65 O-GlcNAcylation and inhibited NFκB p65 phosphorylation at Serine 536, thus promoting IκBα binding to NFκB p65. CONCLUSIONS: There is a reciprocal relationship between O-GlcNAcylation and phosphorylation of NFκB p65, such that increased NFκB p65 O-GlcNAc modification inhibits TNF-α-Induced expression of inflammatory mediators through inhibition of NFκB p65 signaling. These findings provide a mechanistic basis for our previous observations that GlcN and PUGNAc treatments inhibit inflammation and remodeling induced by acute endoluminal arterial injury.

Environmental tobacco smoke furthers progression of diabetic nephropathy.

Clinical studies suggest that smoking is a risk factor in the progression of chronic kidney disease, including diabetic nephropathy. The mechanisms involved are not completely understood. We have previously demonstrated that nicotine, one of the compounds present in large amounts in tobacco, promotes mesangial cell proliferation and fibronectin production. In this study, we hypothesized that exposure to environmental tobacco smoke (ETS) promotes the progression of diabetic nephropathy by increasing the expression of profibrotic cytokines such as transforming growth factor-beta (TGF-ß) and the extracellular matrix proteins fibronectin and collagen IV. Six-week-old diabetic (db/db) mice were divided into 2 groups. The experimental group (n = 12) was exposed to ETS at a concentration of 30 mg/m for 6 hr/d, 5 d/wk for 8 weeks. The control group (n = 8) was exposed to room air. Urine was collected before euthanasia for albumin (enzyme-linked immunosorbent assay) and creatinine measurements (mass spectrometry). After euthanasia, the kidneys were harvested for morphometric analysis and Western blot analysis. Serum was saved for cotinine measurements by enzyme-linked immunosorbent assay. ETS exposure resulted in serum levels of cotinine similar to those found in human smokers. ETS exposure for 8 weeks induced significant mesangial expansion (approximately 50% increase) that was accompanied by concomitant increases in TGF-ß and fibronectin expression (approximately 20%). However, ETS did not modify results in significant changes in urinary albumin excretion. These studies demonstrate that ETS exposure worsens the progression of diabetic nephropathy by increasing the amount of mesangial expansion and that these effects are likely mediated by increased expression of profibrotic cytokines such as TGF-ß.