A. K. Fotheringham

Once daily administration of the SGLT2 inhibitor, empagliflozin, attenuates markers of renal fibrosis without improving albuminuria in diabetic db/db mice.

Blood glucose control is the primary strategy to prevent complications in diabetes. At the onset of kidney disease, therapies that inhibit components of the renin angiotensin system (RAS) are also indicated, but these approaches are not wholly effective. Here, we show that once daily administration of the novel glucose lowering agent, empagliflozin, an SGLT2 inhibitor which targets the kidney to block glucose reabsorption, has the potential to improve kidney disease in type 2 diabetes. In male db/db mice, a 10-week treatment with empagliflozin attenuated the diabetes-induced upregulation of profibrotic gene markers, fibronectin and transforming-growth-factor-beta. Other molecular (collagen IV and connective tissue growth factor) and histological (tubulointerstitial total collagen and glomerular collagen IV accumulation) benefits were seen upon dual therapy with metformin. Albuminuria, urinary markers of tubule damage (kidney injury molecule-1, KIM-1 and neutrophil gelatinase-associated lipocalin, NGAL), kidney growth, and glomerulosclerosis, however, were not improved with empagliflozin or metformin, and plasma and intra-renal renin activity was enhanced with empagliflozin. In this model, blood glucose lowering with empagliflozin attenuated some molecular and histological markers of fibrosis but, as per treatment with metformin, did not provide complete renoprotection. Further research to refine the treatment regimen in type 2 diabetes and nephropathy is warranted.

Targeting advanced glycation endproducts and mitochondrial dysfunction in cardiovascular disease

Cardiovascular disease (CVD) is a leading cause of mortality in the Western World. The development and onset of disease can be attributed to many risk factors including genetic susceptibility, diabetes, obesity and atherosclerosis. Numerous studies highlight the production of advanced glycation endproducts (AGEs) and interaction with their receptor (RAGE) as playing a key pathogenic role. The AGEs-RAGE axis is thought to contribute to a proinflammatory environment inducing cellular dysfunction which cascades towards pathology. Mitochondrial dysfunction concurrently plays a role in these proinflammatory responses presenting excess reactive oxygen species (ROS) production under pathological conditions. This ROS release can exacerbate the production of AGEs fuelling the fire somewhat. However, the AGEs-RAGE axis may influence mitochondrial function independently of inflammation. Therefore instigation of the AGEs-RAGE axis may facilitate spiralling towards pathology on many fronts including CVD development.

Vascular complications in diabetes: old messages, new thoughts

In parallel with the growing diabetes pandemic, there is an increasing burden of micro- and macrovascular complications, occurring in the majority of patients. The identification of a number of synergistic accelerators of disease, providing therapeutic pathways, has stabilised the incidence of complications in most western nations. However, the primary instigators of diabetic complications and, thus, prevention strategies, remain elusive. This has necessitated a refocus on natural history studies, where tissue and plasma samples are sequentially taken to determine when and how disease initiates. In addition, recent Phase III trials, wherein the pleiotropic effects of compounds were arguably as beneficial as their glucose-lowering capacity in slowing the progression of complications, have identified knowledge gaps. Recently the influence of other widely recognised pathological pathways, such as mitochondrial production of reactive oxygen species, has been challenged, highlighting the need for a diverse and robust global research effort to ascertain viable therapeutic targets. Technological advances, such as -omics, high-resolution imaging and computational modelling, are providing opportunities for strengthening and re-evaluating research findings. Newer areas such as epigenetics, energetics and the increasing scrutiny of our synergistic inhabitants, the microbiota, also offer novel targets as biomarkers. Ultimately, however, this field requires concerted lobbying to support all facets of diabetes research.

Rapid genome wide mapping of phosphine resistance loci by a simple regional averaging analysis in the red flour beetle, Tribolium castaneum

BackgroundNext-generation sequencing technology is an important tool for the rapid, genome-wide identification of genetic variations. However, it is difficult to resolve the ‘signal’ of variations of interest and the ‘noise’ of stochastic sequencing and bioinformatic errors in the large datasets that are generated. We report a simple approach to identify regional linkage to a trait that requires only two pools of DNA to be sequenced from progeny of a defined genetic cross (i.e. bulk segregant analysis) at low coverage (<10×) and without parentage assignment of individual SNPs. The analysis relies on regional averaging of pooled SNP frequencies to rapidly scan polymorphisms across the genome for differential regional homozygosity, which is then displayed graphically.ResultsProgeny from defined genetic crosses of Tribolium castaneum (F4 and F19) segregating for the phosphine resistance trait were exposed to phosphine to select for the resistance trait while the remainders were left unexposed. Next generation sequencing was then carried out on the genomic DNA from each pool of selected and unselected insects from each generation. The reads were mapped against the annotated T. castaneum genome from NCBI (v3.0) and analysed for SNP variations. Since it is difficult to accurately call individual SNP frequencies when the depth of sequence coverage is low, variant frequencies were averaged across larger regions. Results from regional SNP frequency averaging identified two loci, tc_rph1 on chromosome 8 and tc_rph2 on chromosome 9, which together are responsible for high level resistance. Identification of the two loci was possible with only 5-7× average coverage of the genome per dataset. These loci were subsequently confirmed by direct SNP marker analysis and fine-scale mapping. Individually, homozygosity of tc_rph1 or tc_rph2 results in only weak resistance to phosphine (estimated at up to 1.5-2.5× and 3-5× respectively), whereas in combination they interact synergistically to provide a high-level resistance >200×. The tc_rph2 resistance allele resulted in a significant fitness cost relative to the wild type allele in unselected beetles over eighteen generations.ConclusionWe have validated the technique of linkage mapping by low-coverage sequencing of progeny from a simple genetic cross. The approach relied on regional averaging of SNP frequencies and was used to successfully identify candidate gene loci for phosphine resistance in T. castaneum. This is a relatively simple and rapid approach to identifying genomic regions associated with traits in defined genetic crosses that does not require any specialised statistical analysis.

Targeted mitochondrial therapy using MitoQ shows equivalent renoprotection to angiotensin converting enzyme inhibition but no combined synergy in diabetes

Mitochondrial dysfunction is a pathological mediator of diabetic kidney disease (DKD). Our objective was to test the mitochondrially targeted agent, MitoQ, alone and in combination with first line therapy for DKD. Intervention therapies (i) vehicle (D); (ii) MitoQ (DMitoQ;0.6 mg/kg/day); (iii) Ramipril (DRam;3 mg/kg/day) or (iv) combination (DCoAd) were administered to male diabetic db/db mice for 12 weeks (n = 11–13/group). Non-diabetic (C) db/m mice were followed concurrently. No therapy altered glycaemic control or body weight. By the study end, both monotherapies improved renal function, decreasing glomerular hyperfiltration and albuminuria. All therapies prevented tubulointerstitial collagen deposition, but glomerular mesangial expansion was unaffected. Renal cortical concentrations of ATP, ADP, AMP, cAMP, creatinine phosphate and ATP:AMP ratio were increased by diabetes and mostly decreased with therapy. A higher creatine phosphate:ATP ratio in diabetic kidney cortices, suggested a decrease in ATP consumption. Diabetes elevated glucose 6-phosphate, fructose 6-phosphate and oxidised (NAD+ and NADP+) and reduced (NADH) nicotinamide dinucleotides, which therapy decreased generally. Diabetes increased mitochondrial oxygen consumption (OCR) at complex II-IV. MitoQ further increased OCR but decreased ATP, suggesting mitochondrial uncoupling as its mechanism of action. MitoQ showed renoprotection equivalent to ramipril but no synergistic benefits of combining these agents were shown.

Perinatal exposure to high dietary advanced glycation end products in transgenic NOD8.3 mice leads to pancreatic beta cell dysfunction

ABSTRACT The contribution of environmental factors to pancreatic islet damage in type 1 diabetes remains poorly understood. In this study, we crossed mice susceptible to type 1 diabetes, where parental male (CD8+ T cells specific for IGRP206-214; NOD8.3) and female (NOD/ShiLt) mice were randomized to a diet either low or high in AGE content and maintained on this diet throughout pregnancy and lactation. After weaning, NOD8.3+ female offspring were identified and maintained on the same parental feeding regimen for until day 28 of life. A low AGE diet, from conception to early postnatal life, decreased circulating AGE concentrations in the female offspring when compared to a high AGE diet. Insulin, proinsulin and glucagon secretion were greater in islets isolated from offspring in the low AGE diet group, which was akin to age matched non-diabetic C57BL/6 mice. Pancreatic islet expression of Ins2 gene was also higher in offspring from the low AGE diet group. Islet expression of glucagon, AGEs and the AGE receptor RAGE, were each reduced in low AGE fed offspring. Islet immune cell infiltration was also decreased in offspring exposed to a low AGE diet. Within pancreatic lymph nodes and spleen, the proportions of CD4+ and CD8+ T cells did not differ between groups. There were no significant changes in body weight, fasting glucose or glycemic hormones. This study demonstrates that reducing exposure to dietary AGEs throughout gestation, lactation and early postnatal life may benefit pancreatic islet secretion and immune infiltration in the type 1 diabetic susceptible mouse strain, NOD8.3.

Ager1 overexpression in glomerular podocytes results in renal disease which is exacerbated by diabetes

Aims: To identify the indications for fistulograms and determine correlation between indication and radiological findings. Background: Dialysis access stenosis is the most common cause of access dysfunction. Clinical monitoring or vascular access surveillance abnormalities prompt a fistulogram. At our institution, fistulogram is primarily used to confirm stenosis. Methods: A retrospective observational study was conducted on 245 fistulograms performed at our institution over a two year period from January 2012 to December 2013. The indication for referral, fistulogram findings, type of fistula and demographic data were obtained. Results: Total of 75.5% (185 of 245) fistulograms performed confirmed stenosis. The most frequent clinical indication was high venous pressures – 18.4% (45 of 245) and the most common surveillance indication was abnormal access flow (Transonic®) – 17.1% (42 of 245). The average age was 57 years, with 35.9% Aboriginal and 28.2% Torres Straight Islander ethnicity. The most common type of vascular access was radiocephalic fistula (49.8%) followed by brachiocephalic fistula (43.3%). Further analysis of variables, using bivariate logistic regression analysis, failed to reveal any significant correlation between indications for referral and finding of stenosis. However, increased venous pressure tends to be associated with stenosis (Odds ratio 2.0, 95% CI = 0.84–4.7, P = 0.12). Both venous hypertension (Odds ratio 0.10, 95% CI = 0.011–1.0, P = 0.052) and development of collaterals (Odds ratio 0.077, 95% CI = 0.0084–0.70, P = 0.023) were associated with negative fistulograms. Conclusions: At our institution, the majority of fistulograms demonstrated access stenosis, based on established referral indications. Both venous hypertension and development of collaterals as referral indications were associated with less likelihood of finding vascular access stenosis in this cohort.Aim: To examine the value of neutrophil-lymphocyte ratio (NLR) as a marker of inflammation and predictor of all-cause mortality in patients with end-stage kidney disease (ESKD). Background: NLR is a marker of systemic inflammation that has been shown to predict mortality in patients with coronary and peripheral vascular disease. In contrast to albumin, NLR is unlikely to be affected by nutritional status. Its prognostic value in ESKD patients is unclear. Methods: We retrospectively reviewed all consecutive haemodialysis patients between January 2007 and December 2011 at a single centre. We recorded patients full blood count and other biochemistry three months after commencement of dialysis. Correlations between NLR and other metabolic and inflammatory markers were evaluated using Pearsons r coefficient. The prognostic value of NLR was tested using Kaplan Meier, univariate and multivariate Cox analyses adjusted for Australian and New Zealand Dialysis and Transplant Registry data. Results: 140 haemodialysis patients were included with median follow-up of 36 months and overall mortality of 41% (58 patients). Neutrophil-lymphocyte ratio was positively correlated with C-reactive protein (r = 0.48, P < 0.01) and negatively correlated with haemoglobin (r = -0.32, P < 0.01) and albumin (r = -0.40, P < 0.01). In Kaplan Meier analysis, NLR (stratified into tertiles) was associated with all-cause mortality (log-rank, P = 0.01). In multivariate Cox analysis, NLR was independently associated with all-cause mortality (HR 1.09, 95% CI 1.01– 1.17 P = 0.03). Other predictors of all-cause mortality in multivariate analysis were low albumin (HR 0.89, 95% CI 0.89–0.94 P < 0.01) and history of cardiovascular disease (HR 2.29, 95% CI 1.25–4.48 P = 0.01). Conclusions: Neutrophil-lymphocyte ratio correlates with other markers of systemic inflammation in ESKD patients and is associated with poor survival. The extent to which other confounding factors affect these results is unknown.Aim: To determine whether: (1) systemic expression of endogenous secretory RAGE (esRAGE) after the induction of diabetes can prevent the development of diabetic nephropathy (DN) in mice with streptozotocin-induced diabetes; (2) the protective effects of esRAGE are attributable to interruption of signaling via the HMGB1receptors (TLR2, TLR4 and RAGE). Background: We have reported that systemic overexpression of esRAGE attenuates diabetic kidney injury. esRAGE is a soluble decoy receptor that can competitively bind ligands for TLRs/RAGE, including HMGB1. Here we test the hypothesis that the protective effects of esRAGE are attributable to interruption of signaling via the HMGB1 receptors (TLR2, TLR4 and RAGE). Methods: DN was induced in WT, TLR4−/− and TLR2−/− mice by intraperitoneal injection of streptozotocin. At 2 weeks after streptozotocin injection, mice received an IP injection of 5 × 1011 vector genome copies (VGC) rAAV encoding either esRAGE or HSA, or saline-control. Samples were collected at week 12 post-induction of diabetes. Results: Diabetic mice that received rAAV-esRAGE, rAAV-HSA or saline developed equivalent degrees of hyperglycaemia. Diabetic WT-mice given rAAVHSA or saline developed significant albuminuria versus non-diabetic WT-mice (ACR309 ± 213 & 313 ± 215 versus 55 ± 10, P < 0.05–0.01), whilst rAAVesRAGE treated-diabetic-mice were protected (118 ± 42, P < 0.05). WT diabetic-mice developed histological damage including glomerular hypertrophy, podocyte injury, macrophage accumulation and interstitial fibrosis. These changes were significantly attenuated in diabetic mice given rAAV-esRAGE versus rAAV-HSA (P < 0.05–0.01).While both TLR2−/− mice and TLR4−/−mice were partially protected against diabetic nephropathy, esRAGE treatment provided additional protection to TLR2−/− mice, but not TLR4−/− mice. A further study of esRAGE treatment in RAGE−/− mice is underway. Conclusions: High-level expression of serum esRAGE after the induction of diabetes provided partial protection against the development of DN in mice with streptozotocin-induced diabetes, which may operate through the TLR4 pathway.

In Db/db mice, Sglt2 inhibition does not improve diabetic nephropathy despite glycaemic improvements

021 CELL BASED THERAPY IN COMBINATION WITH SERELAXIN IS CRITICAL FOR PRESERVATION OF VASCULAR INTEGRITY VIA PROMOTION OF ANGIOGENESIS AND ANASTOMOSIS B HUUSKES1, A PINTO2, C SAMUEL3, S RICARDO1 1Department of Anatomy and Developmental Biology, Monash University, Clayton, Victoria; 2Australian Regenerative Medicine Institute, Monash University, Clayton, Victoria; 3Department of Pharmacology, Monash University, Clayton, Victoria

Delivery of recombinant human soluble receptor for advanced glycation end products delays autoimmune diabetes in the NOD mouse model

Abstracts of 51st EASD Annual Meetings of 51st EASD Annual Meeting OP 01 Insulin analogues: Is newer always better?

Flux of advanced glycation end products across the kidney as a contributor to renal disease in diabetes

and the only basis for predictions of Renal Replacement Therapy (RRT), but there are no systems for monitoring ambulatory CKD in Australia. We have developed a collaborative multidisciplinary platform called CKD.QLD, in which all public renal units in Queensland participate. It includes a registry to characterise CKD patients and their longitudinal course. Website: www.ckdqld.org. Cairns and Hinterland Hospital and Health Service