Aowen Zhuang

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.

Stress in the kidney is the road to pERdition: is endoplasmic reticulum stress a pathogenic mediator of diabetic nephropathy?

The endoplasmic reticulum (ER) is an organelle that primarily functions to synthesise new proteins and degrade old proteins. Owing to the continual and variable nature of protein turnover, protein synthesis is inherently an error-prone process and is therefore tightly regulated. Fortunately, if this balance between synthesis and degradation is perturbed, an intrinsic response, the unfolded protein response (UPR) is activated to restore ER homoeostasis through the action of inositol-requiring protein 1, activating transcription factor 6 and PKR-like ER kinase transmembrane sensors. However, if the UPR is oversaturated and misfolded proteins accumulate, the ER can shift into a cytotoxic response, a physiological phenomenon known as ER stress. The mechanistic pathways of the UPR have been extensively explored; however, the role of this process in such a synthetic organ as the kidney requires further clarification. This review will focus on these aspects and will discuss the role of ER stress in specific resident kidney cells and how this may be integral in the pathogenesis and progression of diabetic nephropathy (DN). Given that diabetes is a perturbed state of protein turnover in most tissues, it is important to understand if ER stress is a secondary or tertiary response to other changes within the diabetic milieu or if it is an independent accelerator of kidney disease. Modulators of ER stress could provide a valuable tool for the treatment of DN and are under active investigation in other contexts.

Regulatory T Cells Prevent Inducible BALT Formation by Dampening Neutrophilic Inflammation

Inducible BALT (iBALT) can amplify pulmonary or systemic inflammatory responses to the benefit or detriment of the host. We took advantage of the age-dependent formation of iBALT to interrogate the underlying mechanisms that give rise to this ectopic, tertiary lymphoid organ. In this study, we show that the reduced propensity for weanling as compared with neonatal mice to form iBALT in response to acute LPS exposure is associated with greater regulatory T cell expansion in the mediastinal lymph nodes. Ab- or transgene-mediated depletion of regulatory T cells in weanling mice upregulated the expression of IL-17A and CXCL9 in the lungs, induced a tissue neutrophilia, and increased the frequency of iBALT to that observed in neonatal mice. Remarkably, neutrophil depletion in neonatal mice decreased the expression of the B cell active cytokines, a proliferation-inducing ligand and IL-21, and attenuated LPS-induced iBALT formation. Taken together, our data implicate a role for neutrophils in lymphoid neogenesis. Neutrophilic inflammation is a common feature of many autoimmune diseases in which iBALT are present and pathogenic, and hence the targeting of neutrophils or their byproducts may serve to ameliorate detrimental lymphoid neogenesis in a variety of disease contexts.

Diabetic kidney disease: a role for advanced glycation end-product receptor 1 (AGE-R1)?

Diabetic patients are postulated to be in a perpetual state of oxidative stress and inflammation at sites where chronic complications occur. The accumulation of AGEs derived from both endogenous and exogenous sources (such as the diet) have been implicated in the development and progression of diabetic complications, particularly nephropathy. There has been some interest in investigating the potential for reducing the AGE burden in chronic disease, through the action of AGE “clearance” receptors, such as the advanced glycation end-product receptor 1 (AGE-R1). Reducing the burden of AGEs has been linked to attenuation of inflammation, slower progression of diabetic complications (in particular vascular and renal complications) and has been shown to extend lifespan. To date, however, there have been no direct investigations into whether AGE-R1 has any role in modulating normal kidney function, or specifically during the development and progression of diabetes. This mini-review will focus on the recent advances in knowledge around the mechanistic function of AGE-R1 and the implications of this for the pathogenesis of diabetic kidney disease.

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.

Increased liver AGEs induce hepatic injury mediated through an OST48 pathway

The protein oligosaccharyltransferase-48 (OST48) is integral to protein N-glycosylation in the endoplasmic reticulum (ER) but is also postulated to act as a membrane localised clearance receptor for advanced glycation end-products (AGE). Hepatic ER stress and AGE accumulation are each implicated in liver injury. Hence the objective of this study was to increase the expression of OST48 and examine the effects on hepatic function and structure. Groups of 8 week old male mice (n = 10–12/group) over-expressing the gene for OST48, dolichyl-diphosphooligosaccharide-protein glycosyltransferase (DDOST+/−), were followed for 24 weeks, while randomised to diets either low or high in AGE content. By week 24 of the study, either increasing OST48 expression or consumption of high AGE diet impaired liver function and modestly increased hepatic fibrosis, but their combination significantly exacerbated liver injury in the absence of steatosis. DDOST+/− mice had increased both portal delivery and accumulation of hepatic AGEs leading to central adiposity, insulin secretory defects, shifted fuel usage to fatty and ketoacids, as well as hepatic glycogen accumulation causing hepatomegaly along with hepatic ER and oxidative stress. This study revealed a novel role of the OST48 and AGE axis in hepatic injury through ER stress, changes in fuel utilisation and glucose intolerance.

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.

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