The protective effect of 1-methyltryptophan isomers in renal ischemia-reperfusion injury is not exclusively dependent on indolamine 2,3-dioxygenase inhibition

BACKGROUND AND PURPOSE: Indolamine 2,3-dioxygenase (IDO), an enzyme that catalyses the metabolism of tryptophan, may play a detrimental role in ischemia-reperfusion injury (IRI). IDO can be inhibited by 1-methyl-tryptophan, which exists in a D (D-MT) or L (L-MT) isomer. These forms show different pharmacological effects besides IDO inhibition. Therefore, we sought to investigate whether these isomers can play a protective role in renal IRI, either IDO-dependent or independent. EXPERIMENTAL APPROACH: We studied the effect of both isomers in a rat renal IRI model with a focus on IDO-dependent and independent effects. KEY RESULTS: Both MT isomers reduced creatinine and BUN levels, with D-MT having a faster onset of action but shorter duration and L-MT a slower onset but longer duration (24 h and 48 h vs 48 h and 96 h reperfusion time). Interestingly, this effect was not exclusively dependent on IDO inhibition, but rather from decreased TLR4 signalling, mimicking changes in renal function. Additionally, L-MT increased the overall survival of rats. Moreover, both MT isomers interfered with TGF-β signalling and epithelial-mesenchymal transition. In order to study the effect of isomers in all mechanisms involved in IRI, a series of in vitro experiments was performed. The isomers affected signalling pathways in NK cells and tubular epithelial cells, as well as in dendritic cells and T cells. CONCLUSION AND IMPLICATIONS: This study shows that both MT isomers have a renoprotective effect after ischemia-reperfusion injury, mostly independent of IDO inhibition, involving mutually different mechanisms. We bring novel findings in the pharmacological properties and mechanism of action of MT isomers, which could become a novel therapeutic target of renal IRI

New genetic loci link adipose and insulin biology to body fat distribution.

Body fat distribution is a heritable trait and a well-established predictor of adverse metabolic outcomes, independent of overall adiposity. To increase our understanding of the genetic basis of body fat distribution and its molecular links to cardiometabolic traits, here we conduct genome-wide association meta-analyses of traits related to waist and hip circumferences in up to 224,459 individuals. We identify 49 loci (33 new) associated with waist-to-hip ratio adjusted for body mass index (BMI), and an additional 19 loci newly associated with related waist and hip circumference measures (P < 5 × 10(-8)). In total, 20 of the 49 waist-to-hip ratio adjusted for BMI loci show significant sexual dimorphism, 19 of which display a stronger effect in women. The identified loci were enriched for genes expressed in adipose tissue and for putative regulatory elements in adipocytes. Pathway analyses implicated adipogenesis, angiogenesis, transcriptional regulation and insulin resistance as processes affecting fat distribution, providing insight into potential pathophysiological mechanisms

Towards graft-specific immune suppression: Gene therapy of the transplanted kidney

Kidney transplantation remains the best therapeutic option for patients with end-stage renal disease. Immunosuppressive therapy has largely resolved the issue of acute transplant rejection. However, because of its systemic nature, immunosuppressive therapy trades off efficacy against side-effects and its chronic use has been associated with severe infections and malignancy. Moreover, long-term survival of renal grafts did not change over the past twenty years. This situation may be improved by using gene therapy as an alternative or add-on strategy to the classic, systemic immune suppression. This review discusses gene therapy approaches in kidney transplantation by addressing the essentials of delivery vectors and by outlining strategies to achieve local immunosupression and allograft-specific tolerance, both in acute rejection and chronic transplant dysfunction. Employing such strategies, local suppression of the immune response and induction of transplantation-specific tolerance have been accomplished in experimental gene therapy. If successful in the clinical setting, gene therapy may (partially) substitute systemic, non-selective immunosuppressive medication, with a major impact on the quality of life and survival of the transplanted patients as well as on the waiting time for receiving a renal graft. (C) 2010 Elsevier B.V. All rights reserved

Targeted renal therapies through microbubbles and ultrasound

Microbubbles and ultrasound enhance the cellular uptake of drugs (including gene constructs) into the kidney. Microbubble induced modifications to the size selectivity of the filtration capacity of the kidney may enable drugs to enter previously inaccessible compartments of the kidney. So far, negative renal side-effects such as capillary bleeding have been reported only in rats, with no apparent damage in larger models such as pigs and rabbits. Although local delivery is accomplished by applying ultrasound only to the target area, efficient delivery using conventional microbubbles has depended on the combined injection of both drugs and microbubbles directly into the renal artery. Conjugation of antibodies to the shell of microbubbles allows for the specific accumulation of microbubbles in the target tissue after intravenous injection. This exciting approach opens new possibilities for both drug delivery and diagnostic ultrasound imaging in the kidney. (C) 2010 Elsevier B.V. All rights reserved

Immune modulation and graft protection by gene therapy in kidney transplantation

Kidney transplantation represents the therapy of choice for many patients with end-stage renal disease. However, the success of renal engraftment is hindered by a number of factors, the most important of which being adverse effects of systemic immunosuppressive therapy, chronic transplant dysfunction and a severe shortage of donor kidneys. Gene therapy approaches may provide valuable strategies in each of these areas. First, gene therapy holds the potential of local therapy, thus circumventing systemic side effects of chronic immunosuppression. Second, chronic transplant dysfunction may be addressed by innovative strategies to induce local immune tolerance, immune suppression and additional graft protecting mechanisms. Third, gene therapy may be instrumental in increasing the quality of the grafts by limiting ischemia-reperfusion injury, especially in non-heart beating donors, thereby expanding the donor pool. In this article, we give an overview of the current state of gene therapy in experimental models of kidney transplantation. (C) 2008 Elsevier B.V. All rights reserved

ULTRASOUND AND MICROBUBBLE-TARGETED DELIVERY OF SMALL INTERFERING RNA INTO PRIMARY ENDOTHELIAL CELLS IS MORE EFFECTIVE THAN DELIVERY OF PLASMID DNA

Ultrasound and microbubble-targeted delivery (UMTD) is a promising non-viral technique for genetic-based therapy. We found that UMTD of small interfering RNA (siRNA) is more effective than delivery of plasmid DNA (pDNA). UMTD (1 MHz, 0.22 MPa) of fluorescently labeled siRNA resulted in 97.9 +/- 1.5% transfected cells, with siRNA localized homogenously in the cytoplasm directly after ultrasound exposure. UMTD of fluorescently labeled pDNA resulted in only 43.0 +/- 4.2% transfected cells, with localization mainly in vesicular structures, co-localizing with endocytosis markers clathrin and caveolin. Delivery of siRNA against GAPDH (glyceraldehyde-3phosphate dehydrogenase) effectively decreased protein levels to 24.3 +/- 7.9% of non-treated controls (p <0.01). In contrast, 24 h after delivery of pDNA encoding GAPDH, no increase in protein levels was detected. Transfection efficiency, verified with red fluorescently labeled pDNA encoding enhanced green fluorescent protein, revealed that of the transfected cells, only 2.0 +/- 0.7% expressed the transgene. In conclusion, the difference in localization between siRNA and pDNA after UMTD is an important determinant of the effectiveness of these genetic-based technologies. (E-mail: [email protected]) (C) 2014 World Federation for Ultrasound in Medicine & Biology

Troubleshooting methods for microarray gene expression analysis in the onset of diabetic kidney disease

<p>Introduction: Microarrays have become the standard technique for discovering new genes involved in the development of (kidney) disease. Diabetic nephropathy is a frequent complication of diabetes and is characterized by renal fibrosis. As the pathways leading to fibrosis are initiated early in diabetes and in the current study, we aimed at identifying genes associated with renal fibrosis in the first week after induction of diabetes in the rat streptozotocin (STZ) model. Methods: Conventional microarray analysis methods comparing gene expression to a common reference are not very suitable for time series as gene lists for all time point are very heterogeneous. We therefore sought an analysis technique that would allow us to easily find genes that we either substantially up or down regulated during the first week of diabetes. In the new method, the normalized expression of individual genes was plotted in time. Subsequently, the area under the curve (AUC) was calculated to quantify the overall level of changes in expression of individual genes. Results: AUCs for all genes were plotted in a histogram showing a normal distribution with a mean of close to 0, indicating no change in expression for the majority of genes. Genes with AUCs outside 3 standard deviations of the mean were considered significantly different from control. Discussion: Using this technique, a total of 290 genes were found to be significantly changed in the first week of diabetes. Data on a subset of genes were confirmed by real-time PCR, indicating the validity of the employed new analysis method. (C) 2013 Elsevier Inc. All rights reserved.</p>

Approaches and methods in gene therapy for kidney disease

Renal gene therapy may offer new strategies to treat diseases of native and transplanted kidneys. Several experimental techniques have been developed and employed using nonviral, viral, and cellular vectors. The most efficient vector for in vivo transfection appears to be adenovirus. Glomeruli, blood vessels, interstitial cells, and pyelum can be transfected with high efficiency. In addition, electroporation and microbubbles with ultrasound, both being enhanced naked plasmid techniques, offer good opportunities. Trapping of mesangial cells into the glomeruli as well as natural targeting of monocytes or macrophages to inflamed kidneys are elegant methods for site-specific delivery of genes. For gene therapy in kidney transplantation, hemagglutinating virus of Japan liposomes are efficient vectors for tubular transfection, whereas enhanced naked plasmid techniques are suitable for glomerular transfection. However, adenovirus offers the best opportunities in a renal transplantation setup because varying parameters of graft perfusion allows targeting of different cell types. In renal grafts, lymphocytes can be used for selective targeting to sites of inflammation. In conclusion, for both in vivo and ex vivo renal transfection, enhanced naked plasmids and adenovirus offer the best perspectives for effective clinical application. Moreover, the development of safer, nonimmunogenic vectors and the large-scale production could make clinical renal gene therapy a realistic possibility for the near future

Metformin Improves Endothelial Function and Reduces Blood Pressure in Diabetic Spontaneously Hypertensive Rats Independent from Glycemia Control: Comparison to Vildagliptin

Metformin confers vascular benefits beyond glycemia control, possibly via pleiotropic effects on endothelial function. In type-1-diabetes-mellitus (T1DM-)patients metformin improved flow-mediated dilation but also increased prostaglandin(PG)-F-2 alpha, a known endothelial-contracting factor. To explain this paradoxical finding we hypothesized that metformin increased endothelial-vasodilator mediators (e.g. NO and EDHF) to an even larger extent. Spontaneously-hypertensive-rats (SHR) display impaired endothelium-dependent relaxation (EDR) involving contractile PGs. EDR was studied in isolated SHR aortas and the involvement of PGs, NO and EDHF assessed. 12-week metformin 300 mg/kg/day improved EDR by up-regulation of NO and particularly EDHF; it also reduced blood pressure and increased plasma sulphide levels (a proxy for H2S, a possible mediator of EDHF). These effects persisted in SHR with streptozotocin (STZ)-induced T1DM. Vildagliptin (10 mg/kg/day), targeting the incretin axis by increasing GLP-1, also reduced blood pressure and improved EDR in SHR aortas, mainly via the inhibition of contractile PGs, but not in STZ-SHR. Neither metformin nor vildagliptin altered blood glucose or HbA(1c). In conclusion, metformin reduced blood pressure and improved EDR in SHR aorta via up-regulation of NO and particularly EDHF, an effect that was independent from glycemia control and maintained during T1DM. A comparison to vildagliptin did not support effects of metformin mediated by GLP-1