Advanced glycation end products cause increased CCN family and extracellular matrix gene expression in the diabetic rodent retina

Aims/hypothesis: Referred to as CCN, the family of growth factors consisting of cystein-rich protein 61 (CYR61, also known as CCN1), connective tissue growth factor (CTGF, also known as CCN2), nephroblastoma overexpressed gene (NOV, also known as CCN3) and WNT1-inducible signalling pathway proteins 1, 2 and 3 (WISP1, -2 and -3; also known as CCN4, -5 and -6) affects cellular growth, differentiation, adhesion and locomotion in wound repair, fibrotic disorders, inflammation and angiogenesis. AGEs formed in the diabetic milieu affect the same processes, leading to diabetic complications including diabetic retinopathy. We hypothesised that pathological effects of AGEs in the diabetic retina are a consequence of AGE-induced alterations in CCN family expression.

Materials and methods: CCN gene expression levels were studied at the mRNA and protein level in retinas of control and diabetic rats using real-time quantitative PCR, western blotting and immunohistochemistry at 6 and 12 weeks of streptozotocin-induced diabetes in the presence or absence of aminoguanidine, an AGE inhibitor. In addition, C57BL/6 mice were repeatedly injected with exogenously formed AGE to establish whether AGE modulate retinal CCN growth factors in vivo.

Results: After 6 weeks of diabetes, Cyr61 expression levels were increased more than threefold. At 12 weeks of diabetes, Ctgf expression levels were increased twofold. Treatment with aminoguanidine inhibited Cyr61 and Ctgf expression in diabetic rats, with reductions of 31 and 36%, respectively, compared with untreated animals. Western blotting showed a twofold increase in CTGF production, which was prevented by aminoguanidine treatment. In mice infused with exogenous AGE, Cyr61 expression increased fourfold and Ctgf expression increased twofold in the retina.

Conclusions/interpolation: CTGF and CYR61 are downstream effectors of AGE in the diabetic retina, implicating them as possible targets for future intervention strategies against the development of diabetic retinopathy.

The effect of increased dietary fruit and vegetable consumption on endothelial activation, inflammation and oxidative stress in hypertensive volunteers

Background and aims

Public health campaigns recommend increased fruit and vegetable (FV) consumption as an effective means of cardiovascular risk reduction. During an 8 week randomised control trial among hypertensive volunteers, we noted significant improvements in endothelium-dependent vasodilatation with increasing FV consumption. Circulating indices of inflammation, endothelial activation and insulin resistance are often employed as alternative surrogates for systemic arterial health. The responses of several such biomarkers to our previously described FV intervention are reported here.
Methods and results

Hypertensive volunteers were recruited from medical outpatient clinics. After a common 4 week run-in period during which FV consumption was limited to 1 portion per day, participants were randomised to 1, 3 or 6 portions daily for 8 weeks. Venous blood samples for biomarker analyses were collected during the pre and post-intervention vascular assessments. A total of 117 volunteers completed the 12 week study. Intervention-related changes in circulating levels of high sensitivity C-reactive protein (hsCRP), soluble intracellular adhesion molecule-1 (sICAM-1), soluble vascular cell adhesion molecule-1 (sVCAM-1), von Willebrand factor (vWF) and plasminogen activator inhibitor-1 (PAI-1) did not differ significantly between FV groups. Similarly, there were no significant between group differences of change in homeostasis model assessment (HOMA) scores.
Conclusions

Despite mediating a significant improvement in acetylcholine induced vasodilatation, increased FV consumption did not affect a calculated measure of insulin resistance or concentrations of the circulating biomarkers measured during this study. Functional indices of arterial health such as endothelium-dependent vasomotion are likely to provide more informative cardiovascular end-points during short-term dietary intervention trials.

Cardiovascular and metabolic abnormalities in the offspring of diabetic pregnancy

Aims/hypothesis. Maternal fuel metabolism is known to exert long range effects on the later development of children of diabetic mothers. Recently cardiovascular disease in adult life has been linked retrospectively with foetal malnutrition. The aim of this study was to identify whether markers for fuel-related cardiovascular programming exist for the offspring of diabetic pregnancy.

The application of NMR to study changes in polar metabolite concentrations in beef longissimus dorsi stored for different periods post mortem

In this study data generated by H-1 NMR spectroscopy were combined with chemometrics to analyse beef samples aged over a 21 day period. In particular, the amino acids, of which 12 were identified were found to increase over the ageing period with samples matured for 3 days having notably lower concentrations than carcasses aged for 21 days. This is believed to be a result of increased proteolysis within the muscle. This novel approach of using high resolution NMR spectrometry to analyse beef samples has not previously been reported and these findings demonstrate the potential of this technique linked with HPLC to be used as a suitable method for profiling meat samples.

Identifying additional patients with diabetic nephropathy using the UK primary care initiative

AIMS:
The aim of this study was to use general practice data to estimate the prevalence of diabetic nephropathy within the registered diabetes patients and examine variation in practice prevalence and management performance since introduction of this initiative.
METHODS:
Reported quality indicators from the Northern Ireland General Practice Quality and Outcomes Framework were analysed for diabetes and diabetic nephropathy prevalence and management in the period 2004-2008. Variation in prevalence at practice level was assessed using multiple linear regression adjusting for age, practice size, deprivation and glycaemic control.
RESULTS:
In 2006-2007, 57,454 (4.1%) adult diabetic patients were registered in the denominator population of 1.4 million compared with 51,923 (3.8%) in 2004-2005 (mean practice range 0.5-7.7%). Diabetic nephropathy prevalence was 15.1 and 11.5%, respectively (8688 and 5955 patients). Documented diabetic nephropathy prevalence showed marked variation across practices (range 0-100%) and was significantly negatively correlated with diabetes list size, albumin creatinine ratio testing rates and renin-angiotensin-aldosterone system blockade use and positively correlated with exception reporting rates. Specifically, for every increase in 100 diabetic patients to a register, documented diabetic nephropathy prevalence reduced by 40% (P=0.003). On the positive side, median albumin-creatinine ratio testing rates doubled to 82% compared with figures in the pre-Framework era.
CONCLUSIONS:
Implementation of the Northern Ireland General Practice Quality and Outcomes Framework has positively benefitted testing for diabetic nephropathy and increased numbers of detected patients in a short space of time. Large variation in diabetic nephropathy prevalence remains and is associated with diabetes registry size, screening and treatment practices, suggesting that understanding this variation may help detect and better manage diabetic nephropathy.

Association analysis of Notch pathway signalling genes in diabetic nephropathy

Several studies have provided compelling evidence implicating the Notch signalling pathway in diabetic nephropathy. Co-regulation of Notch signalling pathway genes with GREM1 has recently been demonstrated and several genes involved in the Notch pathway are differentially expressed in kidney biopsies from individuals with diabetic nephropathy. We assessed single-nucleotide polymorphisms (SNPs; n = 42) in four of these key genes (JAG1, HES1, NOTCH3 and ADAM10) for association with diabetic nephropathy using a case-control design.
Tag SNPs and potentially functional SNPs were genotyped using Sequenom or Taqman technologies in a total of 1371 individuals with type 1 diabetes (668 patients with nephropathy and 703 controls without nephropathy). Patients and controls were white and recruited from the UK and Ireland. Association analyses were performed using PLINK (http://pngu.mgh.harvard.edu/similar to purcell/plink/) and haplotype frequencies in patients and controls were compared. Adjustment for multiple testing was performed by permutation testing.
In analyses stratified by centre, we identified six SNPs, rs8708 and rs11699674 (JAG1), rs10423702 and rs1548555 (NOTCH3), rs2054096 and rs8027998 (ADAM10) as being associated with diabetic nephropathy before, but not after, adjustment for multiple testing. Haplotype and subgroup analysis according to duration of diabetes also failed to find an association with diabetic nephropathy.
Our results suggest that common variants in JAG1, HES1, NOTCH3 and ADAM10 are not strongly associated with diabetic nephropathy in type 1 diabetes among white individuals. Our findings, however, cannot entirely exclude these genes from involvement in the pathogenesis of diabetic nephropathy.

Effect of antioxidants and ACE inhibition on chemical modification of proteins and progression of nephropathy in the streptozotocin diabetic rat

Aims/hypothesis. This study was designed to determine whether inhibition of formation of AGE and advanced lipoxidation end-products (ALE) is a mechanism of action common to a diverse group of therapeutic agents that limit the progress of diabetic nephropathy. We compared the effects of the ACE inhibitor enalapril, the antioxidant vitamin E, the thiol compound lipoic acid, and the AGE/ALE inhibitor pyridoxamine on the formation of AGE/ALE and protection against nephropathy in streptozotocin diabetic rats.

Identification of a Second Kindred with Familial Hypocalciuric Hypercalcemia Type 3 (FHH3) Narrows Localization to a

Context: Familial hypocalciuric hypercalcemia (FHH) is a genetically heterogenous disorder that consists of three defined types, FHH1, FHH2, and FHH3 whose chromosomal locations are 3q21.1, 19p, and 19q13, respectively. FHH1, caused by mutations of the calcium-sensing receptor (CASR), occurs in more than 65% of patients, whereas the abnormalities underlying FHH2 and FHH3, which have each been described in single North American kindreds, are unknown.

Allelic depletion of grem1 attenuates diabetic kidney disease.

OBJECTIVE: Gremlin (grem1) is an antagonist of the bone morphogenetic protein family that plays a key role in limb bud development and kidney formation. There is a growing appreciation that altered grem1 expression may regulate the homeostatic constraints on damage responses in diseases such as diabetic nephropathy. RESEARCH DESIGN AND METHODS: Here we explored whether knockout mice heterozygous for grem1 gene deletion (grem1(+/-)) exhibit protection from the progression of diabetic kidney disease in a streptozotocin-induced model of type 1 diabetes. RESULTS: A marked elevation in grem1 expression was detected in the kidneys and particularly in kidney tubules of diabetic wild-type mice compared with those of littermate controls. In contrast, diabetic grem1(+/-) mice displayed a significant attenuation in grem1 expression at 6 months of diabetes compared with that in age- and sex-matched wild-type controls. Whereas the onset and induction of diabetes were similar between grem1(+/-) and wild-type mice, several indicators of diabetes-associated kidney damage such as increased glomerular basement membrane thickening and microalbuminuria were attenuated in grem1(+/-) mice compared with those in wild-type controls. Markers of renal damage such as fibronectin and connective tissue growth factor were elevated in diabetic wild-type but not in grem1(+/-) kidneys. Levels of pSmad1/5/8 decreased in wild-type but not in grem1(+/-) diabetic kidneys, suggesting that bone morphogenetic protein signaling may be maintained in the absence of grem1. CONCLUSIONS: These data identify grem1 as a potential modifier of renal injury in the context of diabetic kidney disease.

Müller glial dysfunction during diabetic retinopathy in rats is linked to accumulation of advanced glycation end-products and advanced lipoxidation end-products.

Aims/hypothesis: The impact of AGEs and advanced lipoxidation end-products (ALEs) on neuronal and Müller glial dysfunction in the diabetic retina is not well understood. We therefore sought to identify dysfunction of the retinal Müller glia during diabetes and to determine whether inhibition of AGEs/ALEs can prevent it.

Methods: Sprague-Dawley rats were divided into three groups: (1) non-diabetic; (2) untreated streptozotocin-induced diabetic; and (3) diabetic treated with the AGE/ALE inhibitor pyridoxamine for the duration of diabetes. Rats were killed and their retinas were evaluated for neuroglial pathology. Results: AGEs and ALEs accumulated at higher levels in diabetic retinas than in controls (p<0.001). AGE/ALE immunoreactivity was significantly diminished by pyridoxamine treatment of diabetic rats. Diabetes was also associated with the up-regulation of the oxidative stress marker haemoxygenase-1 and the induction of glial fibrillary acidic protein production in Müller glia (p<0.001). Pyridoxamine treatment of diabetic rats had a significant beneficial effect on both variables (p<0.001). Diabetes also significantly altered the normal localisation of the potassium inwardly rectifying channel Kir4.1 and the water channel aquaporin 4 to the Müller glia end-feet interacting with retinal capillaries. These abnormalities were prevented by pyridoxamine treatment.

Conclusions/interpretation: While it is established that AGE/ALE formation in the retina during diabetes is linked to microvascular dysfunction, this study suggests that these pathogenic adducts also play a role in Müller glial dysfunction.