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Diabetic nephropathy (DN) is the leading cause of end-stage kidney disease. Besides glycemic and blood pressure control, environmental factors such as cigarette smoking (CS) adversely affect the progression of DN. The effects of CS on DN progression have been attributed to combustion-generated molecules without consideration to the role of nicotine (NIC), responsible for the addictive properties of both CS and electronic cigarettes (ECs). Podocytes are essential to preserve the structure and function of the glomerular filtration barrier, and strong evidence indicates that early podocyte loss promotes DN progression. We performed experiments in human podocytes and in a mouse model of diabetes that develops nephropathy resembling human DN. We determined that NIC binding to podocytes in concentrations achieved with CS and ECs activated NADPH oxidase, which sets in motion a dysfunctional molecular network integrated by cyclooxygenase 2, known to induce podocyte injury; downregulation of AMP-activated protein kinase, important for maintaining cellular energy stores and antioxidation; and upregulation of CD36, which increased lipid uptake and promoted apoptosis. In diabetic mice, NIC increased proteinuria, a recognized marker of chronic kidney disease progression, accompanied by reduced glomerular podocyte synaptopodin, a crucial stabilizer of the podocyte cytoskeleton, and increased fibronectin expression. This novel study critically implicates NIC itself as a contributor to DN progression in CS and EC users. NEW & NOTEWORTHY In this study, we demonstrate that nicotine increases the production of reactive oxygen species, increases cyclooxygenase-2 expression, and upregulates Cd36 while inducing downregulation of AMP-activated protein kinase. In vivo nicotine increases proteinuria and fibronectin expression in diabetic mice. This study demonstrates that effects of nicotine on podocytes are responsible, at least in part, for the deleterious effects of smoking in the progression of chronic kidney disease, including diabetic nephropathy. [ABSTRACT FROM AUTHOR]

Heterozygosity for human polycystic kidney and hepatic disease 1 (PKHD1) mutations was recently associated with cystic liver disease and radiographic findings resembling medullary sponge kidney (MSK). However, the relevance of these associations has been tempered by a lack of cystic liver or renal disease in heterozygous mice carrying Pkhd1 gene trap or exon deletions. To determine whether heterozygosity for a smaller Pkhd1 defect can trigger cystic renal disease in mice, we generated and characterized mice with the predicted truncating Pkhd1C642** mutation in a region corresponding to the middle of exon 20 cluster of five truncating human mutations (between PKHD1G617fs and PKHD1G644*). Mouse heterozygotes or homozygotes for the Pkhd1C642** mutation did not have noticeable liver or renal abnormalities on magnetic resonance images during their first weeks of life. However, when aged to ~1.5 yr, the Pkhd1C642** heterozygotes developed prominent cystic liver changes; tissue analyses revealed biliary cysts and increased number of bile ducts without signs of congenital hepatic fibrosis-like portal field inflammation and fibrosis that was seen in Pkhd1C642** homozygotes. Interestingly, aged female Pkhd1C642** heterozygotes, as well as homozygotes, developed radiographic changes resembling MSK. However, these changes correspond to proximal tubule ectasia, not an MSK-associated collecting duct ectasia. In summary, by demonstrating that cystic liver and kidney abnormalities are triggered by heterozygosity for the Pkhd1C642** mutation, we provide important validation for relevant human association studies. Together, these investigations indicate that PKHD1 mutation heterozygosity (predicted frequency 1 in 70 individuals) is an important underlying cause of cystic liver disorders and MSK-like manifestations in a human population. [ABSTRACT FROM AUTHOR]

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-β 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 saltsensitive hypertension. [ABSTRACT FROM AUTHOR]

The article tests the hypothesis that erythroblastosis virus E26 oncogen homolog-1 (ETS-1) is a common mediator of the renal proinflammatory profibrotic effects of Angiotensis II (Ang II). Studies suggest that ETS-1 is a common mediator of the proinflammatory and profibrotic effects of Ang II-induced hypertensive renal damage. Such mediation may result in the development of novel strategies in the treatment and prevention of end-organ injury in hypertension.

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-β 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The transcription factor ETS-1 regulates the expression of several growth factors, chemokines and cytokines. We have recently shown that ETS-1 is a mediator of the pro-inflammatory and pro-fibrotic effects of Angiotensin II in the kidney (HTN ’12). Herein, we tested the hypothesis that the renal expression of ETS-1 is increased in the Dahl salt-sensitive (DS) rat, a paradigm of salt-sensitive hypertension in humans, and that ETS-1 mediates renal injury in this model of hypertension. DS rats (n=6 per group) were fed a normal salt diet (0.5% NaCl, NS) or a high salt diet (4% NaCl, HS) for 4 weeks. Four additional groups (n=6 per group) on HS diet received: ETS-1 dominant negative peptide (HS/DN, 10mg/kg/day) to block ETS-1, a control ETS-1 mutant peptide (HS/MU, 10mg /kg/day), the AT1 receptor blocker Candesartan (HS/ARB 10 mg/kg/day) or a combination of DN and ARB (HS/DN/ARB). HS rats had a ~ 3 fold increase in the cortical expression of ETS-1 as assessed by western blot (WB). Treatment with DN, MU, ARB or DN/ARB resulted in small and non-significant reductions in BP as compared to HS. (Table). Compared with LS, HS rats had increased proteinuria (Bio-Rad), higher glomerular injury score (GIS), increased fibronectin expression (WB) and urinary TGF-β (ELISA) that were improved by DN but not by MU (table). ARB reduced proteinuria and GIS (table). Treatment with DN/ARB resulted in further improvements in renal injury as compared to DN and ARB (table).In conclusion we have demonstrated that hypertensive DS rats have increased cortical expression of ETS-1 and that blockade of ETS-1 improves renal injury in this model of hypertension. [ABSTRACT FROM AUTHOR]

Tobacco smoking is associated with accelerated progression of chronic kidney disease of different etiologies including diabetes and hypertension. However, the mechanisms involved are not well understood. We have previously reported that nicotine, a biologically active compound present in high concentrations in tobacco, induces cell proliferation and fibronectin production in mesangial cells which are prevented by ERK1/2 inhibition (AJP’05). In these studies we determined whether rat mesangial cells (MC) express nicotine receptors and characterized the signaling pathways that lead to ERK1/2 phosphorylation in response to nicotine. MC were grown in DMEM with 15% FBS in the presence of 0.4 mg/ml G418 and starved for 24 hours in DMEM without FBS before treatment. We first demonstrated that MC are endowed with several nicotinic Ach receptor (nAChR) subunits including α2-7 and β1-4 as assessed by western blot. Treatment of rat MC with nicotine at 10-7M caused a time-dependent ERK1/2 phosphorylation which peaked after 10 min of stimulation( N=3). Several protein kinase inhibitors were then used to identify the upstream kinases that mediate nicotine-induced ERK1/2 phosphorylation. The calcium/calmodulin-dependent protein kinase II (CaMKII) inhibitor KN93 (10-7M) decreased the ERK1/2 phosphorylation level by ∼57% (0.41 of 0.95) as compared to nicotine. The PKC inhibitor Go6983 at 10-9M, the PKA inhibitor H89 (10-8 M) and the EGFR inhibitor AG 1478 (10-7 M) also inhibited ERK1/2 phosphorylation by 60% (0.38 of 0.95), 48% (0.63 of 1.20) and 68% (0.40 of 1.23) respectively as compared to nicotine. Given the role of the nicotine receptors as agonist-regulated Ca2+ channels, we determined the effects of Ca2+ channel blockade on nicotine induced ERK1/2 phosphorylation. Treatment of MC with the calcium channel Verapamil (10-9 M) resulted in 33% (0.49 of 0.73) inhibition of ERK1/2 phosphorylation as compared to nicotine. In summary, we have determined in these studies that rat MC are endowed with several nAChR subunits and that ERK1/2 phosphorylation in response to nicotine requires CaMK II, PKA, PKC and EGFR. In addition, we have demonstrated that these effects require Ca2+ consistent with the role of the nAChR as agonist-regulated Ca2+ channels in MC. [ABSTRACT FROM AUTHOR]

Airway microbial dysbiosis is associated with subsequent bronchopulmonary dysplasia (BPD) development in very preterm infants. However, the relationship of airway microbiome in normal pulmonary development has not been defined. To better understand the role of the airway microbiome, we compared normal and abnormal alveolar and pulmonary vascular development in mice with or without a microbiome. We hypothesized that the lungs of germ-free (GF) mice would have an exaggerated phenotypic response to hyperoxia compared with non-germ-free (NGF) mice. With the use of a novel gnotobiotic hyperoxia chamber, GF and NGF mice were exposed to either normoxia or hyperoxia. Alveolar morphometry, pulmonary mechanics, echocardiograms, inflammatory markers, and measures of pulmonary hypertension were studied. GF and NGF mice in normoxia showed no difference, whereas GF mice in hyperoxia showed protected lung structure and mechanics and decreased markers of inflammation compared with NGF mice. We speculate that an increase in abundance of pathogenic bacteria in NGF mice may play a role in BPD pathogenesis by regulating the proinflammatory signaling and neutrophilic inflammation in lungs. Manipulation of the airway microbiome may be a potential therapeutic intervention in BPD and other lung diseases.

Hyperoxia-induced oxidant stress contributes to the pathogenesis of bronchopulmonary dysplasia (BPD) in preterm infants. Mitochondrial functional differences due to mitochondrial DNA (mtDNA) variations are important modifiers of oxidant stress responses. The objective of this study was to determine whether mtDNA variation independently modifies lung development and mechanical dysfunction in newborn mice exposed to hyperoxia. Newborn C57BL6 wild type (C57 n /C57 mt , C57WT) and C3H/HeN wild type (C3H n /C3H mt , C3HWT) mice and novel Mitochondrial-nuclear eXchange (MNX) strains with nuclear DNA (nDNA) from their parent strain and mtDNA from the other-C57MNX (C57 n /C3H mt ) and C3HMNX (C3H n /C57 mt )-were exposed to 21% or 85% O 2 from birth to postnatal day 14 (P14). Lung mechanics and histopathology were examined on P15. Neonatal mouse lung fibroblast (NMLF) bioenergetics and mitochondrial superoxide (O 2 - ) generation were measured. Pulmonary resistance and mitochondrial O 2 - generation were increased while alveolarization, compliance, and NMLF basal and maximal oxygen consumption rate were decreased in hyperoxia-exposed C57WT mice (C57 n /C57 mt ) versus C57MNX mice (C57 n /C3H mt ) and in hyperoxia-exposed C3HMNX mice (C3H n /C57 mt ) versus C3HWT (C3H n /C3H mt ) mice. Our study suggests that neonatal C57 mtDNA-carrying strains have increased hyperoxia-induced hypoalveolarization, pulmonary mechanical dysfunction, and mitochondrial bioenergetic and redox dysfunction versus C3H mtDNA strains. Therefore, mtDNA haplogroup variation-induced differences in mitochondrial function could modify neonatal alveolar development and BPD susceptibility.

Heterozygosity for human polycystic kidney and hepatic disease 1 ( PKHD1) mutations was recently associated with cystic liver disease and radiographic findings resembling medullary sponge kidney (MSK). However, the relevance of these associations has been tempered by a lack of cystic liver or renal disease in heterozygous mice carrying Pkhd1 gene trap or exon deletions. To determine whether heterozygosity for a smaller Pkhd1 defect can trigger cystic renal disease in mice, we generated and characterized mice with the predicted truncating Pkhd1 C642* mutation in a region corresponding to the middle of exon 20 cluster of five truncating human mutations (between PKHD1 G617fs and PKHD1 G644* ). Mouse heterozygotes or homozygotes for the Pkhd1 C642* mutation did not have noticeable liver or renal abnormalities on magnetic resonance images during their first weeks of life. However, when aged to ~1.5 yr, the Pkhd1 C642* heterozygotes developed prominent cystic liver changes; tissue analyses revealed biliary cysts and increased number of bile ducts without signs of congenital hepatic fibrosis-like portal field inflammation and fibrosis that was seen in Pkhd1 C642* homozygotes. Interestingly, aged female Pkhd1 C642* heterozygotes, as well as homozygotes, developed radiographic changes resembling MSK. However, these changes correspond to proximal tubule ectasia, not an MSK-associated collecting duct ectasia. In summary, by demonstrating that cystic liver and kidney abnormalities are triggered by heterozygosity for the Pkhd1 C642* mutation, we provide important validation for relevant human association studies. Together, these investigations indicate that PKHD1 mutation heterozygosity (predicted frequency 1 in 70 individuals) is an important underlying cause of cystic liver disorders and MSK-like manifestations in a human population.

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.