Differential effects of superoxide on luminal and basolateral Na+/H+ exchange in the thick ascending limb

Superoxide (O2-) increases Na+ reabsorption in the thick ascending limb (THAL) by enhancing Na/K/2Cl cotransport. However, the effects of O2- on other THAL transporters, such as Na(+)/H+ exchangers, are unknown. We hypothesized that O2- stimulates Na(+)/H+ exchange in the THAL. We assessed total Na(+)/H+ exchange activity by measuring recovery of intracellular pH (pH(i)) after acid loading in isolated perfused THALs before and after adding xanthine oxidase (XO) and hypoxanthine (HX). We found that XO and HX decreased total pH(i) recovery rate from 0.26 +/- 0.05 to 0.21 +/- 0.04 pH units/min (P < 0.05), and this net inhibition decreased steady-state pH(i) from 7.52 to 7.37. Because THALs have different Na(+)/H+ exchanger isoforms on the luminal and basolateral membrane, we tested the effects of xanthine oxidase and hypoxanthine on luminal and basolateral Na(+)/H+ exchange by adding dimethylamiloride to either the bath or lumen. Xanthine oxidase and hypoxanthine increased luminal Na(+)/H+ exchange from 3.5 +/- 0.8 to 6.7 +/- 1.4 pmol.min(-1).mm(-1) (P < 0.01) but decreased basolateral Na(+)/H+ exchange from 10.8 +/- 1.8 to 6.8 +/- 1.1 pmol.min(-1).mm(-1) (P < 0.007). To ascertain whether these effects were caused by O2- or H2O2, we examined the ability of tempol, a superoxide dismutase mimetic, to block these effects. In the presence of tempol, xanthine oxidase and hypoxanthine had no effect on luminal or basolateral Na(+)/H+ exchange. We conclude that O2- inhibits basolateral and stimulates luminal Na(+)/H+ exchangers, perhaps because different isoforms are expressed on each membrane. Inhibition of basolateral Na(+)/H+ exchange may enhance stimulation of luminal Na(+)/H+ exchange by providing additional protons to be extruded across the luminal membrane. Together, the effects of O2- on Na(+)/H+ exchange may increase net HCO3- reabsorption by the THAL.     

A model wheat cultivar for transformation to improve resistance to Fusarium Head Blight

Fusarium head blight (FHB), caused primarily by Fusarium graminearum, is a major disease problem in wheat (Triticum aestivum). Genetic engineering holds significant potential to enhance FHB resistance in wheat. Due to the requirement of screening for FHB resistance on flowers at anthesis, the number of screens carried out in a year is limited. Our objective was to evaluate the feasibility of using the rapid-maturing dwarf wheat cultivar Apogee as an alternative genotype for transgenic FHB resistance research. Our transformation efficiency (number of transgenic plants/number of embryos) for Apogee was 1.33%. Apogee was also found to exhibit high FHB susceptibility and reached anthesis within 4 weeks. Interestingly, microsatellite marker haplotype analysis of the chromosome 3BS FHB resistant quantitative trait locus (QTL) region indicated that this region maybe deleted in Apogee. Our results indicate that Apogee is particularly well suited for accelerating transgenic FHB resistance research and transgenic wheat research in general. C.A. Mackintosh and D.F. Garvin contributed equally to the article and should be considered co-first authors     

Estradiol increases VEGF in human breast studied by whole-tissue culture

Sex steroid exposure constitutes a risk factor for breast cancer, but little is known about the effects of sex steroids on the normal breast, largely because of the lack of convenient models. We have developed a method of culturing normal breast tissue ex vivo. We have applied this method to investigate the effects of estradiol and progesterone on the key angiogenic mediator, vascular endothelial growth factor (VEGF), in the breast. Whole breast tissue was obtained from routine reduction mammoplasty. Tissue biopsies were cultured in vitro for 1-3 weeks, and the expression of luminal cytokeratin 18 was determined by immunohistochemistry. As an application, tissue biopsies were treated in vitro for 1 week with or without estradiol or estradiol and progesterone. Estrogen receptor, progesterone receptor, and Ki-67 were analyzed, and VEGF levels were examined by quantitative immunoassay and immunohistochemistry. Whole breast tissue was cultured ex vivo for 1 week with preserved morphology. Increased detachment of the luminal epithelium was observed after 2 weeks. Estradiol increased extracellular levels of VEGF in normal breast tissue biopsy medium. The addition of progesterone had neither stimulatory nor inhibitory effects on secreted VEGF. The method of whole breast tissue culturing thus provide a means by which to explore the biology of normal breast tissue. Our results suggest that estradiol exerts pro-angiogenic effects in normal breast by increasing levels of biologically active VEGF.     

Application of single nucleotide polymorphisms to non-model species: a technical review

Single nucleotide polymorphisms (SNPs) have gained wide use in humans and model species and are becoming the marker of choice for applications in other species. Technology that was developed for work in model species may provide useful tools for SNP discovery and genotyping in non-model organisms. However, SNP discovery can be expensive, labour intensive, and introduce ascertainment bias. In addition, the most efficient approaches to SNP discovery will depend on the research questions that the markers are to resolve as well as the focal species. We discuss advantages and disadvantages of several past and recent technologies for SNP discovery and genotyping and summarize a variety of SNP discovery and genotyping studies in ecology and evolution.     

Angiotensin II Stimulates Thick Ascending Limb Superoxide Production via Protein Kinase Cα-dependent NADPH Oxidase Activation

Angiotensin II (Ang II) stimulates thick ascending limb (TAL) O production, but the receptor(s) and signaling mechanism(s) involved are unknown. The effect of Ang II on O is generally attributed to the AT₁ receptor. In some cells, Ang II stimulates protein kinase C (PKC), whose α isoform (PKCα) can activate NADPH oxidase. We hypothesized that in TALs, Ang II stimulates O via AT₁ and PKCα-dependent NADPH oxidase activation. In rat TALs, 1 nm Ang II stimulated O from 0.76 ± 0.17 to 1.97 ± 0.21 nmol/min/mg (p < 0.001). An AT₁ antagonist blocked the stimulatory effect of Ang II on O (0.87 ± 0.25 nmol/min/mg; p < 0.006), whereas an AT₂ antagonist had no effect (2.16 ± 0.133 nmol/min/mg; p < 0.05 versus vehicle). Apocynin, an NADPH oxidase inhibitor, blocked Ang II-stimulated O by 90% (p < 0.01). Ang II failed to stimulate O in TALs from p47^phox−/− mice (p < 0.02). Monitored by fluorescence resonance energy transfer, Ang II increased PKC activity from 0.02 ± 0.03 to 0.13 ± 0.02 arbitrary units (p < 0.03). A general PKC inhibitor, GF109203X, blocked the effect of Ang II on O (1.47 ± 0.21 versus 2.72 ± 0.47 nmol/min/mg with Ang II alone; p < 0.03). A PKCα- and β-selective inhibitor, Gö6976, also blocked the stimulatory effect of Ang II on O (0.59 ± 0.15 versus 2.05 ± 0.28 nmol/min/mg with Ang II alone; p < 0.001). To distinguish between PKCα and PKCβ, we used tubules expressing dominant-negative PKCα or -β. In control TALs, Ang II stimulated O by 2.17 ± 0.44 nmol/min/mg (p < 0.011). In tubules expressing dominant-negative PKCα, Ang II failed to stimulate O (change: −0.30 ± 0.27 nmol/min/mg). In tubules expressing dominant-negative PKCβ1, Ang II stimulated O by 2.08 ± 0.69 nmol/min/mg (p < 0.002). We conclude that Ang II stimulates TAL O production via activation of AT₁ receptors and PKCα-dependent NADPH oxidase.     

Cardiovascular risk management in rheumatoid arthritis: are we still waiting for the first step?

Rheumatoid arthritis (RA) is associated with a similar cardiovascular risk to that in diabetes, and therefore cardiovascular risk management (CV-RM) - that is, identification and treatment of cardiovascular risk factors (CRFs) - is mandatory. However, whether and to what extent this is done in daily clinical practice is unknown. In a retrospective cohort investigation, CV-RM was therefore compared between rheumatologists and primary care physicians (PCPs). Remarkably, CRFs in RA were less frequently identified and managed by rheumatologists in comparison with PCPs. In addition, PCPs assessed CRFs less frequently in RA than in diabetes. Obviously, there is a clear need for improvement of CV-RM in RA and this should be a joint effort from the rheumatologist and the PCP.Patients with rheumatoid arthritis (RA) have an increased cardiovascular (CV) risk that appears similar to that in diabetes. This observation highlights the significant CV burden in RA. In 1999, the American Diabetes Association and the American Heart Association published a statement for prevention of CV disease in diabetes. Since then, the CV risk in diabetes has been substantially lower than in earlier decades. Given the increased CV risk in RA, screening, identification of cardiovascular risk factors (CRFs) and cardiovascular risk management (CV-RM) are also highly needed as recommended by the European League Against Rheumatism (EULAR). The increased risk in RA is attributed to systemic inflammation as well as increased prevalence of CRFs. Hence, we should aim for tight disease control and control of CRFs.Presently unknown is whether and to what extent CV-RM is translated into clinical practice. In a retrospective cohort-comprising 251 patients with RA, 251 patients with diabetes, and 251 general population individuals-Desai and colleagues therefore investigated the identification and management of CRFs by rheumatologists and primary care physicians (PCPs) [1]. RA patients had to be registered at the University of Michigan Health System for at least 12 months between June 2007 and April 2012 and had been evaluated both by their rheumatologist as well as the PCP. CRFs of interest were smoking, exercise, weight, blood pressure, lipid profile, and fasting blood glucose.In RA, PCPs identified and managed most CRFs more frequently than rheumatologists. Secondly, identification of CRFs by rheumatologists in RA patients with elevated C-reactive protein levels was not different as compared with those with normal C-reactive protein levels. A third important observation was that PCPs identified and managed CRFs more frequently in patients with diabetes, followed by general population individuals and least often in RA patients. These striking results raise several issues.First, it is hard to believe that the largely absent CV-RM by rheumatologists is explained by under-recognition because the increased CV risk in RA must presently be well known among rheumatologists. A large amount of literature on this topic has been published over the last decade. Additionally, the necessity to screen, identify, and manage CRFs is incorporated into training programmes for rheumatology residents [2]. Against this background, it is important to realise that there is a lag time between the publication of the EULAR guideline and its actual implementation (that is, the guideline was published in 2010 [3] while the current study started in 2007). In other words, CV-RM in today''s clinical practice might have been improved, but not yet recognised.Second, that the CV risk in RA is related to the inflammatory burden is well known. Nevertheless, the present study did not indicate that there is more attention for CV-RM by rheumatologists in patients with a higher inflammatory load.Third, undertreatment of the increased CV risk in RA by PCPs might be explained by under-recognition because CRFs were assessed more frequently in diabetes in comparison with RA.The EULAR guidelines recommend screening and identification of CRFs in all RA patients, and, if indicated according to CV risk-prediction charts, adequate management. As accurate assessment of CV risk depends on RA characteristics, the EULAR favoured individualising risk assessment. Hence, a risk multiplication factor of 1.5 should be used in the presence of two of the following criteria: disease duration >10 years, rheumatoid factor, and/or anti-cyclic citrullinated peptide positivity or the presence of extra-articular manifestations. However, alternative approaches have been suggested - for example, increasing the age of an RA patient by 10 years to obtain a more precise CV risk estimate or to use other risk scores. Perhaps this lack of an RA-specific CV risk-prediction model hampers CV-RM implementation. Obviously, this discussion can only be solved by developing a RA-specific CV risk-prediction model, but this will take several years to complete.One may obviously argue that, due to its retrospective design, the strength of the conclusions of Desai and colleagues may be limited; however, they are in line with other recently published literature and thus confirm extending evidence that CV-RM is poorly conducted in RA, both by rheumatologists and PCPs. Another argument against CV-RM in RA is that we should wait until trials have been conducted that demonstrate the efficacy of statins and antihypertensive agents in RA. However, it will be (many) years before specific risk models are available and withholding cardiopreventive drugs that are very likely to work also in our high-risk population is unethical. Moreover, it is important to realise that, due to the decreased incidence of CV events in the last decades, CV prevention trials are nowadays very difficult to conduct. For instance, the TRACE-RA study [4] - a large placebo-controlled double-blind primary CV prevention trial in RA with atorvastatin - was stopped prematurely owing to the very low number of CV events that occurred.Altogether, the study from Desai and colleagues provides three important clues for improvement of CV-RM in RA. First, more education is urgently needed for both rheumatologists and PCPs. Second, it is important to realise that the contribution of higher prevalence CRFs in RA is one side of the coin, but the other side is effective suppression of the inflammation. The latter is a clear task for the rheumatologist. Third, CV care of a RA patient should be a joint effort by the rheumatologist and the PCP, and they should collaborate and agree on who performs the screening, identification, and, if required, management of CRFs.     

Heme oxygenase metabolites inhibit tubuloglomerular feedback in vivo

Tubuloglomerular feedback (TGF) is a renal autoregulatory mechanism that constricts the afferent arteriole in response to increases in distal NaCl. Heme oxygenases (HO-1 and HO-2) release carbon monoxide (CO) and biliverdin, which may help control renal function. We showed in vitro that HO products inhibit TGF; however, we do not know whether this also occurs in vivo or the mechanism(s) involved. We hypothesized that in vivo HO-1 and HO-2 in the nephron inhibit TGF via release of CO and biliverdin. We first performed laser capture microdissection followed by real-time PCR and found that both HO-1 and HO-2 are expressed in the macula densa. We next performed micropuncture experiments in vivo on individual rat nephrons, adding different compounds to the perfusate, and found that an HO inhibitor, stannous mesoporphyrin (SnMP), potentiated TGF (P < 0.05, SnMP vs. control). The CO-releasing molecule (CORM)-3 partially inhibited TGF at 50 μmol/l (P < 0.01, CORM-3 vs. control) and blocked it completely at higher doses. A soluble guanylyl cyclase (sGC) inhibitor, LY83583, blocked the inhibitory effect of CORM-3 on TGF. Biliverdin also partially inhibited TGF (P < 0.01, biliverdin vs. control), most likely attributable to decreased superoxide (O(2)(-)) because biliverdin was rendered ineffective by tempol, a O(2)(-) dismutase mimetic. We concluded that HO-1 and HO-2 in the nephron inhibit TGF by releasing CO and biliverdin. The inhibitory effect of CO on TGF is mediated by the sGC/cGMP signaling pathway, whereas biliverdin probably acts by reducing O(2)(-).     

Morphine self-administration in the rat during adjuvant-induced arthritis

Rats injected with Freund's adjuvant develop a syndrome resembling human rheumatoid arthritis complete with paw swelling, edema and persistent pain. At the onset of pain, arthritic rats and their pain-free littermate controls (vehicle injection) were allowed to self-administer intravenous morphine (5.0 mg/kg/injection) in a 24 hr/day schedule. Self-injected morphine appeared to provide analgesia in arthritic rats as demonstrated by a decreased sensitivity to applied tail pressure. Arthritic rats self-inject significantly less morphine than pain-free animals. Injection of indomethacin, which alleviates the pain and inflammation of the adjuvant-induced disease, reduces, at least initially, morphine self-injection in the arthritic but not pain-free animals. As the adjuvant-induced inflammation and pain dissipated, arthritic rats rapidly began to increase opioid intake. The presence of persistent pain apparently reduces the addictive properties of morphine.