Novel Genetic Determinants of Diabetic Kidney Disease

Diabetic kidney disease (DKD) is a devastating diabetes complication, with known heritability not fully revealed by previous genetics studies. We performed the largest genome-wide association study of type 1 DKD to date, in a 13-cohort consortium of 15,590 individuals of European ancestry genotyped on the Illumina HumanCoreExome Beadchip, which allows exploration of coding variation in addition to genomic markers.

As prior work has shown that different characterizations of the DKD phenotype highlight distinct genetic associations, we investigated a spectrum of DKD definitions based on proteinuria and renal function criteria. Controls were DKD-free after a minimum of 15 years diabetes duration; cases had diabetes for at least 10 years prior to DKD diagnosis. We also performed a quantitative trait analysis of estimated glomerular filtration rate in all participants.

Our top finding was a missense mutation in COL4A3, rs55703767 (Asp326Tyr); the minor allele is common in Europeans (20%) and East Asians (13%) but not Africans (2%). This SNP had a genome-wide significant association with traditionally defined DKD (macroalbuminuria or end-stage renal disease [ESRD], (OR= 0.79, P=1.9×10-9), and a suggestive association with macroalbuminuria (OR= 0.79, P=1.6×10-6) and ESRD (OR= 0.79, P=4.5×10-5) individually. Though its PolyPhen score is 0.3 (benign), this SNP has been implicated as a splice site disruptor.

The COL4A3 gene encodes the alpha 3 subunit of Type IV collagen, the major structural component of basement membranes. Pathogenic mutations in COL4A3 have been identified in thin basement membrane nephropathy, familial focal segmental glomerulosclerosis, and Alport syndrome. A proxy (r2=0.6) for rs55703767 had no significant associations in the CKDGen consortium, suggesting its pathogenicity occurs solely in the setting of hyperglycemia.

By significantly increasing sample size we have discovered a novel locus underlying DKD risk, paving the way for better understanding of pathology, prevention, and treatment.

Getting to the heart of cardiac remodeling; how collagen subtypes may contribute to phenotype

The objective of this study was to investigate the nature and biomechanical properties of collagen fibers within the human myocardium. Targeting cardiac interstitial abnormalities will likely become a major focus of future preventative strategies with regard to the management of cardiac dysfunction. Current knowledge regarding the component structures of myocardial collagen networks is limited, further delineation of which will require application of more innovative technologies. We applied a novel methodology involving combined confocal laser scanning and atomic force microscopy to investigate myocardial collagen within ex-vivo right atrial tissue from 10 patients undergoing elective coronary bypass surgery. Immuno-fluorescent co-staining revealed discrete collagen I and III fibers. During single fiber deformation, overall median values of stiffness recorded in collagen III were 37±16% lower than in collagen I [p<0.001]. On fiber retraction, collagen I exhibited greater degrees of elastic recoil [p<0.001; relative percentage increase in elastic recoil 7±3%] and less energy dissipation than collagen III [p<0.001; relative percentage increase in work recovered 7±2%]. In atrial biopsies taken from patients in permanent atrial fibrillation (n=5) versus sinus rhythm (n=5), stiffness of both collagen fiber subtypes was augmented (p<0.008). Myocardial fibrillar collagen fibers organize in a discrete manner and possess distinct biomechanical differences; specifically, collagen I fibers exhibit relatively higher stiffness, contrasting with higher susceptibility to plastic deformation and less energy efficiency on deformation with collagen III fibers. Augmented stiffness of both collagen fiber subtypes in tissue samples from patients with atrial fibrillation compared to those in sinus rhythm are consistent with recent published findings of increased collagen cross-linking in this setting.

Natural history of markers of collagen turnover in patients with early diastolic dysfunction and impact of eplerenone

OBJECTIVES: This study was designed to evaluate the impact of eplerenone on collagen turnover in preserved systolic function heart failure (HFPSF).

BACKGROUND: Despite growing interest in abnormal collagen metabolism as a feature of HFPSF with diastolic dysfunction, the natural history of markers of collagen turnover and the impact of selective aldosterone antagonism on this natural history remains unknown.

METHODS: We evaluated 44 patients with HFPSF, randomly assigned to control (n = 20) or eplerenone 25 mg daily (n = 24) for 6 months, increased to 50 mg daily from 6 to 12 months. Serum markers of collagen turnover and inflammation were analyzed at baseline and at 6 and 12 months and included pro-collagen type-I and -III aminoterminal peptides, matrix metalloproteinase type-2, interleukin-6 and -8, and tumor necrosis factor-alpha. Doppler-echocardiographic assessment of diastolic filling indexes and tissue Doppler analyses were also obtained.

RESULTS: The mean age of the patients was 80 +/- 7.8 years; 46% were male; 64% were receiving an angiotensin-converting enzyme inhibitor, 34% an angiotensin-II receptor blocker, and 68% were receiving beta-blocker therapy. Pro-collagen type-III and -I aminoterminal peptides, matrix metalloproteinase type-2, interleukin-6 and -8, and tumor necrosis factor-alpha increased with time in the control group. Eplerenone treatment had no significant impact on any biomarker at 6 months but attenuated the increase in pro-collagen type-III aminoterminal peptide at 12 months (p = 0.006). Eplerenone therapy was associated with modest effects on diastolic function without any impact on clinical variables or brain natriuretic peptide.

CONCLUSIONS: This study demonstrates progressive increases in markers of collagen turnover and inflammation in HFPSF with diastolic dysfunction. Despite high background utilization of renin-angiotensin-aldosterone modulators, eplerenone therapy prevents a progressive increase in pro-collagen type-III aminoterminal peptide and may have a role in management of this disease. (The Effect of Eplerenone and Atorvastatin on Markers of Collagen Turnover in Diastolic Heart Failure; NCT00505336).

Hypoxia-induced DNA hypermethylation in human pulmonary fibroblasts is associated with Thy-1 promoter methylation and the development of a pro-fibrotic phenotype

BACKGROUND: Pulmonary fibrosis is a debilitating and lethal disease with no effective treatment options. Understanding the pathological processes at play will direct the application of novel therapeutic avenues. Hypoxia has been implicated in the pathogenesis of pulmonary fibrosis yet the precise mechanism by which it contributes to disease progression remains to be fully elucidated. It has been shown that chronic hypoxia can alter DNA methylation patterns in tumour-derived cell lines. This epigenetic alteration can induce changes in cellular phenotype with promoter methylation being associated with gene silencing. Of particular relevance to idiopathic pulmonary fibrosis (IPF) is the observation that Thy-1 promoter methylation is associated with a myofibroblast phenotype where loss of Thy-1 occurs alongside increased alpha smooth muscle actin (α-SMA) expression. The initial aim of this study was to determine whether hypoxia regulates DNA methylation in normal human lung fibroblasts (CCD19Lu). As it has been reported that hypoxia suppresses Thy-1 expression during lung development we also studied the effect of hypoxia on Thy-1 promoter methylation and gene expression.

METHODS: CCD19Lu were grown for up to 8 days in hypoxia and assessed for global changes in DNA methylation using flow cytometry. Real-time PCR was used to quantify expression of Thy-1, α-SMA, collagen I and III. Genomic DNA was bisulphite treated and methylation specific PCR (MSPCR) was used to examine the methylation status of the Thy-1 promoter.

RESULTS: Significant global hypermethylation was detected in hypoxic fibroblasts relative to normoxic controls and was accompanied by increased expression of myofibroblast markers. Thy-1 mRNA expression was suppressed in hypoxic cells, which was restored with the demethylating agent 5-aza-2'-deoxycytidine. MSPCR revealed that Thy-1 became methylated following fibroblast exposure to 1% O2.

CONCLUSION: These data suggest that global and gene-specific changes in DNA methylation may play an important role in fibroblast function in hypoxia.

Use of Galleria mellonella as a model organism to study Legionella pneumophila infection.

Legionella pneumophila, the causative agent of a severe pneumonia named Legionnaires' disease, is an important human pathogen that infects and replicates within alveolar macrophages. Its virulence depends on the Dot/Icm type IV secretion system (T4SS), which is essential to establish a replication permissive vacuole known as the Legionella containing vacuole (LCV). L. pneumophila infection can be modeled in mice however most mouse strains are not permissive, leading to the search for novel infection models. We have recently shown that the larvae of the wax moth Galleria mellonella are suitable for investigation of L. pneumophila infection. G. mellonella is increasingly used as an infection model for human pathogens and a good correlation exists between virulence of several bacterial species in the insect and in mammalian models. A key component of the larvae's immune defenses are hemocytes, professional phagocytes, which take up and destroy invaders. L. pneumophila is able to infect, form a LCV and replicate within these cells. Here we demonstrate protocols for analyzing L. pneumophila virulence in the G. mellonella model, including how to grow infectious L. pneumophila, pretreat the larvae with inhibitors, infect the larvae and how to extract infected cells for quantification and immunofluorescence microscopy. We also describe how to quantify bacterial replication and fitness in competition assays. These approaches allow for the rapid screening of mutants to determine factors important in L. pneumophila virulence, describing a new tool to aid our understanding of this complex pathogen.

Legionella pneumophila secretes a mitochondrial carrier protein during infection

The Mitochondrial Carrier Family (MCF) is a signature group of integral membrane proteins that transport metabolites across the mitochondrial inner membrane in eukaryotes. MCF proteins are characterized by six transmembrane segments that assemble to form a highly-selective channel for metabolite transport. We discovered a novel MCF member, termed Legionellanucleotide carrier Protein (LncP), encoded in the genome of Legionella pneumophila, the causative agent of Legionnaire's disease. LncP was secreted via the bacterial Dot/Icm type IV secretion system into macrophages and assembled in the mitochondrial inner membrane. In a yeast cellular system, LncP induced a dominant-negative phenotype that was rescued by deleting an endogenous ATP carrier. Substrate transport studies on purified LncP reconstituted in liposomes revealed that it catalyzes unidirectional transport and exchange of ATP transport across membranes, thereby supporting a role for LncP as an ATP transporter. A hidden Markov model revealed further MCF proteins in the intracellular pathogens, Legionella longbeachae and Neorickettsia sennetsu, thereby challenging the notion that MCF proteins exist exclusively in eukaryotic organisms.

Subcellular localization of legionella Dot/Icm effectors

The translocation of effector proteins by the Dot/Icm type IV secretion system is central to the ability of Legionella pneumophila to persist and replicate within eukaryotic cells. The subcellular localization of translocated Dot/Icm proteins in host cells provides insight into their function. Through co-staining with host cell markers, effector proteins may be localized to specific subcellular compartments and membranes, which frequently reflects their host cell target and mechanism of action. In this chapter, we describe protocols to (1) localize effector proteins within cells by ectopic expression using green fluorescent protein fusions and (2) localize effector proteins within infected cells using epitope-tagged effector proteins and immuno-fluorescence microscopy.

LtpD is a novel legionella pneumophila effector that binds phosphatidylinositol 3-phosphate and inositol monophosphatase IMPA1

The Dot/Icm type IV secretion system (T4SS) of Legionella pneumophila is crucial for the pathogen to survive in protozoa and cause human disease. Although more than 275 effector proteins are delivered into the host cell by the T4SS, the function of the majority is unknown. Here we have characterized the Dot/Icm effector LtpD. During infection, LtpD localized to the cytoplasmic face of the membrane of the Legionella-containing vacuole (LCV). In A549 lung epithelial cells, ectopically expressed LtpD localized to large vesicular structures that contained markers of endosomal compartments. Systematic analysis of LtpD fragments identified an internal 17-kDa fragment, LtpD_471-626, which was essential for targeting ectopically expressed LtpD to vesicular structures and for the association of translocated LtpD with the LCV. LtpD_471-626 bound directly to phosphatidylinositol 3-phosphate [PtdIns(3)P] in vitro and colocalized with the PtdIns(3)P markers FYVE and SetA in cotransfected cells. LtpD was also found to bind the host cell enzyme inositol (myo)-1 (or 4)-monophosphatase 1, an important phosphatase involved in phosphoinositide production. Analysis of the role of LtpD in infection showed that LtpD is involved in bacterial replication in THP-1 macrophages, the larvae of Galleria mellonella, and mouse lungs. Together, these data suggest that LtpD is a novel phosphoinositide- binding L. pneumophila effector that has a role in intracellular bacterial replication. © 2013, American Society for Microbiology.

Long-term statin therapy in patients with systolic heart failure and normal cholesterol:effects on elevated serum markers of collagen turnover, inflammation, and B-type natriuretic peptide

BACKGROUND: The role of statin therapy in heart failure (HF) is unclear. The amino-terminal propeptide of procollagen type III (PIIINP) predicts outcome in HF, and yet there are conflicting reports of statin therapy effects on PIIINP.

OBJECTIVES: This study determined whether there was an increase in serum markers of inflammation, fibrosis (including PIIINP), and B-type natriuretic peptide (BNP) in patients with systolic HF and normal total cholesterol and determined the effects of long-term treatment with atorvastatin on these markers.

METHODS: Fifty-six white patients with systolic HF and normal cholesterol levels (age 72 [13] years; 68% male; body mass index 27.0 [7.3] kg/m(2); ejection fraction 35 [13]%; 46% with history of smoking) were randomly allocated to atorvastatin treatment for 6 months, titrated to 40 mg/d (A group) or not (C group). Age- and/or sex-matched subjects without HF (N group) were also recruited. Biomarkers were measured at baseline (all groups) and 6 months (A and C groups).

RESULTS: Serum markers of collagen turnover, inflammation, and BNP were significantly elevated in HF patients compared with normal participants (all P < 0.05). There were correlations between these markers in HF patients but not in normal subjects. Atorvastatin treatment for 6 months caused a significant reduction in the following biomarkers compared with baseline: BNP, from median (interquartile range) 268 (190-441) pg/mL to 185 (144-344) pg/mL; high-sensitivity C-reactive protein (hs-CRP), from 5.26 (1.95 -9.29) mg/L to 3.70 (2.34-6.81) mg/L; and PIIINP, from 4.65 (1.86) to 4.09 (1.25) pg/mL (all P < 0.05 baseline vs 6 months). Between-group differences were significant for PIIINP only (P = 0.027). There was a positive interaction between atorvastatin effects and baseline hs-CRP and PIIINP (P < 0.01).

CONCLUSIONS: Long-term statin therapy reduced PIIINP in this small, selected HF population with elevated baseline levels. Further evaluation of statin therapy in the management of HF patients with elevated PIIINP is warranted.

Extracellular matrix sub-types and mechanical stretch impact human cardiac fibroblast responses to transforming growth factor beta

Understanding the impact of extracellular matrix sub-types and mechanical stretch on cardiac fibroblast activity is required to help unravel the pathophysiology of myocardial fibrotic diseases. Therefore, the purpose of this study was to investigate pro-fibrotic responses of primary human cardiac fibroblast cells exposed to different extracellular matrix components, including collagen sub-types I, III, IV, VI and laminin. The impact of mechanical cyclical stretch and treatment with transforming growth factor beta 1 (TGFβ1) on collagen 1, collagen 3 and alpha smooth muscle actin mRNA expression on different matrices was assessed using quantitative real-time PCR. Our results revealed that all of the matrices studied not only affected the expression of pro-fibrotic genes in primary human cardiac fibroblast cells at rest but also affected their response to TGFβ1. In addition, differential cellular responses to mechanical cyclical stretch were observed depending on the type of matrix the cells were adhered to. These findings may give insight into the impact of selective pathological deposition of extracellular matrix proteins within different disease states and how these could impact the fibrotic environment.

Combining histology, stable isotope analysis and ZooMS collagen fingerprinting to investigate the taphonomic history and dietary behaviour of extinct giant tortoises from the Mare aux Songes deposit on Mauritius

Taphonomic research of bones can provide additional insight into a site's formation and development, the burial environment and ongoing post-mortem processes. A total of 30 tortoise (Cylindraspis) femur bone samples from the Mare aux Songes site (Mauritius)were studied histologically, assessing parameters such as presence and type of microbial alteration, inclusions, staining/infiltrations, the degree of microcracking and birefringence. The absence of microbial attack in the 4200 year old Mare aux Songes bones suggests the animals rapidly entered the soil whole-bodied and were sealed anoxically, although they suffered frombiological and chemical degradation (i.e. pyrite formation/oxidation, mineral dissolution and staining) related to changes in the site's hydrology. Additionally, carbon and nitrogen stable isotopeswere analysed to obtain information on the animals' feeding behaviour. The results show narrowly distributed δ13C ratios, indicating a terrestrial C3 plant-based diet, combined with a wide range in δ15N ratios. This is most likely related to the tortoises' drought-adaptive ability to change their metabolic processes, which can affect the δ15N ratios. Furthermore, ZooMS collagen fingerprinting analysis successfully identified two tortoise species (C. triserrata and C. inepta) in the bone assemblage,which,when combined with stable isotope data, revealed significantly different δ15N ratios between the two tortoise species. As climatic changes around this period resulted in increased aridity in the Mascarene Islands, this could explain the extremely elevated δ15N ratio in our dataset. The endemic fauna was able to endure the climatic changes 4200 years ago, although human arrival in the 17th century changed the original habitat to such an extent that it resulted in the extinction of several species. Fortunately we are still able to study these extinct tortoises due to the beneficial conditions of their burial environment, resulting in excellent bone preservation.