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Understanding functional interactions between G protein-coupled receptors is of great physiological and pathophysiological importance. Heteromerization provides one important potential mechanism for such interaction between different signalling pathways via macromolecular complex formation. Previous studies suggested a functional interplay between angiotensin II receptor type 1 (AT1) and Chemokine (C-C motif) Receptor 2 (CCR2). However the molecular mechanisms are not understood. We investigated AT1-CCR2 functional interaction in vitro using bioluminescence resonance energy transfer in HEK293 cells and in vivo using subtotal-nephrectomized rats as a well-established model for chronic kidney disease. Our data revealed functional heteromers of these receptors resulting in CCR2-Gαi1 coupling being sensitive to AT1 activation, as well as apparent enhanced β-arrestin2 recruitment with agonist co-stimulation that is synergistically reversed by combined antagonist treatment. Moreover, we present in vivo findings where combined treatment with AT1- and CCR2-selective inhibitors was synergistically beneficial in terms of decreasing proteinuria, reducing podocyte loss and preventing renal injury independent of blood pressure in the subtotal-nephrectomized rat model. Our findings further support a role for G protein-coupled receptor functional heteromerization in pathophysiology and provide insights into previous observations indicating the importance of AT1-CCR2 functional interaction in inflammation, renal and hypertensive disorders.

Diabetic retinopathy features inflammation as well as injury to glial cells and the microvasculature, which are influenced by hypertension and overactivity of the renin-angiotensin system. FT011 is an anti-inflammatory and anti-fibrotic agent that has been reported to attenuate organ damage in diabetic rats with cardiomyopathy and nephropathy. However, the potential therapeutic utility of FT011 for diabetic retinopathy has not been evaluated. We hypothesized that FT011 would attenuate retinopathy in diabetic Ren-2 rats, which exhibit hypertension due to an overactive extra-renal renin-angiotensin system. Diabetic rats were studied for 8 and 32 weeks and received intravitreal injections of FT011 (50 μM) or vehicle (0.9% NaCl). Comparisons were to age-matched controls. In the 8-week study, retinal inflammation was examined by quantitating vascular leukocyte adherence, microglial/macrophage density and the expression of inflammatory mediators. Macroglial Müller cells, which exhibit a pro-inflammatory and pro-angiogenic phenotype in diabetes, were evaluated in the 8-week study as well as in culture following exposure to hyperglycaemia and FT011 (10, 30, 100 μM) for 72 hours. In the 32-week study, severe retinal vasculopathy was examined by quantitating acellular capillaries and extracellular matrix proteins. In diabetic rats, FT011 reduced retinal leukostasis, microglial density and mRNA levels of intercellular adhesion molecule-1 (ICAM-1). In Müller cells, FT011 reduced diabetes-induced gliosis and vascular endothelial growth factor (VEGF) immunolabeling and the hyperglycaemic-induced increase in ICAM-1, monocyte chemoattractant protein-1, CCL20, cytokine-induced neutrophil chemoattractant-1, VEGF and IL-6. Late intervention with FT011 reduced acellular capillaries and the elevated mRNA levels of collagen IV and fibronectin in diabetic rats. In conclusion, the protective effects of FT011 in cardiorenal disease extend to key elements of diabetic retinopathy and highlight its potential as a treatment approach.

The progressive decline of renal function in chronic kidney disease (CKD) is characterized by both disruption of the microvascular architecture and the accumulation of fibrotic matrix. One angiogenic pathway recently identified as playing an essential role in renal vascular development is the stromal cell-derived factor-1α (SDF-1)/CXCR4 pathway. Because similar developmental processes may be recapitulated in the disease setting, we hypothesized that the SDF-1/CXCR4 system would regulate microvascular health in CKD. Expression of CXCR4 was observed to be increased in the kidneys of subtotally nephrectomized (SNx) rats and in biopsies from patients with secondary focal segmental glomerulosclerosis (FSGS), a rodent model and human correlate both characterized by aberration of the renal microvessels. A reno-protective role for local SDF-1/CXCR4 signaling was indicated by i) CXCR4-dependent glomerular eNOS activation following acute SDF-1 administration; and ii) acceleration of renal function decline, capillary loss and fibrosis in SNx rats treated with chronic CXCR4 blockade. In contrast to the upregulation of CXCR4, SDF-1 transcript levels were decreased in SNx rat kidneys as well as in renal fibroblasts exposed to the pro-fibrotic cytokine transforming growth factor β (TGF-β), the latter effect being attenuated by histone deacetylase inhibition. Increased renal SDF-1 expression was, however, observed following the treatment of SNx rats with the ACE inhibitor, perindopril. Collectively, these observations indicate that local SDF-1/CXCR4 signaling functions to preserve microvascular integrity and prevent renal fibrosis. Augmentation of this pathway, either purposefully or serendipitously with either novel or existing therapies, may attenuate renal decline in CKD.

BACKGROUND: Obesity is associated with diastolic dysfunction, lower maximal myocardial blood flow, impaired myocardial metabolism and increased risk of heart failure. We examined the association between obesity, left ventricular filling pressure and myocardial structure. METHODS: We performed histological analysis of non-ischemic myocardium from 57 patients (46 men and 11 women) undergoing coronary artery bypass graft surgery who did not have previous cardiac surgery, myocardial infarction, heart failure, atrial fibrillation or loop diuretic therapy. RESULTS: Non-obese (body mass index, BMI, ≤ 30 kg/m(2), n=33) and obese patients (BMI >30 kg/m(2), n=24) did not differ with respect to myocardial total, interstitial or perivascular fibrosis, arteriolar dimensions, or cardiomyocyte width. Obese patients had lower capillary length density (1145 ± 239, mean ± SD, vs. 1371 ± 333 mm/mm(3), P=0.007) and higher diffusion radius (16.9 ± 1.5 vs. 15.6 ± 2.0 μm, P=0.012), in comparison with non-obese patients. However, the diffusion radius/cardiomyocyte width ratio of obese patients (0.73 ± 0.11 μm/μm) was not significantly different from that of non-obese patients (0.71 ± 0.11 μm/μm), suggesting that differences in cardiomyocyte width explained in part the differences in capillary length density and diffusion radius between non-obese and obese patients. Increased BMI was associated with increased pulmonary capillary wedge pressure (PCWP, P

BACKGROUND: Both ageing and hypertension are known risk factors for atrial fibrillation (AF) although the pathophysiological contribution or interaction of the individual factors remains poorly understood. Here we aim to delineate the arrhythmogenic atrial substrate in mature spontaneously hypertensive rats (SHR). METHODS: SHR were studied at 12 and 15 months of age (n = 8 per group) together with equal numbers of age-matched normotensive Wistar-Kyoto control rats (WKY). Electrophysiologic study was performed on superfused isolated right and left atrial preparations using a custom built high-density multiple-electrode array to determine effective refractory periods (ERP), atrial conduction and atrial arrhythmia inducibility. Tissue specimens were harvested for structural analysis. RESULTS: COMPARED TO WKY CONTROLS, THE SHR DEMONSTRATED: Higher systolic blood pressure (p

An accelerated progressive decline in renal function is a frequent accompaniment of myocardial infarction (MI). Indoxyl sulfate (IS), a uremic toxin that accumulates from the early stages of chronic kidney disease (CKD), is contributory to both renal and cardiac fibrosis. IS levels can be reduced by administration of the oral adsorbent AST-120, which has been shown to ameliorate pathological renal and cardiac fibrosis in moderate to severe CKD. However, the cardiorenal effect of AST-120 on less severe renal dysfunction in the post-MI setting has not previously been well studied. MI-induced Sprague-Dawley rats were randomized to receive either AST-120 (MI+AST-120) or were untreated (MI+Vehicle) for 16 weeks. Serum IS levels were measured at baseline, 8 and 16 weeks. Echocardiography and glomerular filtration rate (GFR) were assessed prior to sacrifice. Renal and cardiac tissues were assessed for pathological changes using histological and immunohistochemical methods, Western blot analysis and real-time PCR. Compared with sham, MI+Vehicle animals had a significant reduction in left ventricular ejection fraction (by 42%, p

Cardiovascular death commonly occurs in patients with chronic kidney disease. Indoxyl sulfate (IS), a uremic toxin, has been demonstrated in vitro as a contributory factor in cardiac fibrosis, a typical pathological finding in uremic cardiomyopathy. This study aimed to determine if cardiac fibrosis is reversible by lowering serum IS levels using an oral charcoal adsorbent, AST-120. Subtotal-nephrectomized (5/6-STNx) Sprague-Dawley rats were randomized to receive either AST-120 (AST-120, n=13) or no treatment (vehicle, n=17) for 12 weeks. Sham operated rats (n=12) were used as controls. Early left ventricular (LV) diastolic dysfunction was demonstrated by an increase in peak velocity of atrial filling [A and A' waves] and a decrease of E/A and E'/A' ratios obtained by echocardiography. This was accompanied by a 4.5-fold increase in serum IS (p

Locally-active growth factors have been implicated in the pathogenesis of many diseases in which organ fibrosis is a characteristic feature. In the setting of chronic kidney disease (CKD), two such pro-fibrotic factors, transforming growth factor-β (TGF-β) and platelet-derived growth factor (PDGF) have emerged as lead potential targets for intervention. Given the incomplete organ protection afforded by blocking the actions of TGF-β or PDGF individually, we sought to determine whether an agent that inhibited the actions of both may have broader effects in ameliorating the key structural and functional abnormalities of CKD.Accordingly, we studied the effects of a recently described, small molecule anti-fibrotic drug, 3-methoxy-4-propargyloxycinnamoyl anthranilate (FT011, Fibrotech Therapeutics, Australia), which should have these effects.In the in vitro setting, FT011 inhibited both TGF-β1 and PDGF-BB induced collagen production as well as PDGF-BB-mediated mesangial proliferation. Consistent with these in vitro actions, when studied in a robust model of non-diabetic kidney disease, the 5/6 nephrectomised rat, FT011 attenuated the decline in GFR, proteinuria and glomerulosclerosis (p

BACKGROUND: Heart failure is associated with abnormalities of myocardial structure, and plasma levels of the advanced glycation end-product (AGE) N(ε)-(carboxymethyl)lysine (CML) correlate with the severity and prognosis of heart failure. Aging is associated with diastolic dysfunction and increased risk of heart failure, and we investigated the hypothesis that diastolic dysfunction of aging humans is associated with altered myocardial structure and plasma AGE levels. METHODS: We performed histological analysis of non-ischemic left ventricular myocardial biopsies and measured plasma levels of the AGEs CML and low molecular weight fluorophores (LMWFs) in 26 men undergoing coronary artery bypass graft surgery who had transthoracic echocardiography before surgery. None had previous cardiac surgery, myocardial infarction, atrial fibrillation, or heart failure. RESULTS: The patients were aged 43-78 years and increasing age was associated with echocardiographic indices of diastolic dysfunction, with higher mitral Doppler flow velocity A wave (r = 0.50, P = 0.02), lower mitral E/A wave ratio (r = 0.64, P = 0.001), longer mitral valve deceleration time (r = 0.42, P = 0.03) and lower early diastolic peak velocity of the mitral septal annulus, e' (r = 0.55, P = 0.008). However, neither mitral E/A ratio nor mitral septal e' was correlated with myocardial total, interstitial or perivascular fibrosis (picrosirius red), immunostaining for collagens I and III, CML, and receptor for AGEs (RAGE), cardiomyocyte width, capillary length density, diffusion radius or arteriolar dimensions. Plasma AGE levels were not associated with age. However, plasma CML levels were associated with E/A ratio (r = 0.44, P = 0.04) and e' (r = 0.51, P = 0.02) and LMWF levels were associated with E/A ratio (r = 0.49, P = 0.02). Moreover, the mitral E/A ratio remained correlated with plasma LMWF levels in all patients (P = 0.04) and the mitral septal e' remained correlated with plasma CML levels in non-diabetic patients (P = 0.007) when age was a covariate. CONCLUSIONS: Diastolic dysfunction of aging was independent of myocardial structure but was associated with plasma AGE levels.

BackgroundDiabetic cardiomyopathy (DCM) is an increasingly recognized cause of chronic heart failure amongst diabetic patients. Both increased reactive oxygen species (ROS) generation and impaired ROS scavenging have been implicated in the pathogenesis of hyperglycemia-induced left ventricular dysfunction, cardiac fibrosis, apoptosis and hypertrophy. We hypothesized that 3',4'-dihydroxyflavonol (DiOHF), a small highly lipid soluble synthetic flavonol, may prevent DCM by scavenging ROS, thus preventing ROS-induced cardiac damage.Methodology/principal findingsSix week old homozygous Ren-2 rats were randomized to receive either streptozotocin or citrate buffer, then further randomized to receive either DiOHF (1 mg/kg/day) by oral gavage or vehicle for six weeks. Cardiac function was assessed via echocardiography and left ventricular cardiac catheterization before the animals were sacrificed and hearts removed for histological and molecular analyses. Diabetic Ren-2 rats showed evidence of diastolic dysfunction with prolonged deceleration time, reduced E/A ratio, and increased slope of end-diastolic pressure volume relationship (EDPVR) in association with marked interstitial fibrosis and oxidative stress (all PConclusionsInhibition of ROS production and action by DiOHF improved diastolic function and reduced myocyte hypertrophy as well as collagen deposition. These findings suggest the potential clinical utility of antioxidative compounds such as flavonols in the prevention of diabetes-associated cardiac dysfunction.

Most forms of chronic kidney disease are characterized by progressive renal and cardiac fibrosis leading to dysfunction. Preliminary evidence suggests that various bone marrow-derived cell populations have antifibrotic effects. In exploring the therapeutic potential of bone marrow derived cells in chronic cardio-renal disease, we examined the anti-fibrotic effects of bone marrow-derived culture modified cells (CMCs) and stromal cells (SCs).In vitro, CMC-conditioned medium, but not SC-conditioned medium, inhibited fibroblast collagen production and cell signalling in response to transforming growth factor-beta. The antifibrotic effects of CMCs and SCs were then evaluated in the 5/6 nephrectomy model of chronic cardio-renal disease. While intravascular infusion of 10(6) SCs had no effect, 10(6) CMCs reduced renal fibrosis compared to saline in the glomeruli (glomerulosclerosis index: 0.8+/-0.1 v 1.9+/-0.2 arbitrary units) and the tubulointersitium (% area type IV collagen: 1.2+/-0.3 v 8.4+/-2.0, p

Associations between chronic diabetes complications and mitochondrial dysfunction represent a subject of major importance, given the diabetes pandemic and high personal and socioeconomic costs of diabetes and its complications. Modelling diabetes complications in inbred laboratory animals is challenging due to incomplete recapitulation of human features, but offer mechanistic insights and preclinical testing. As mitochondrial-based oxidative stress is implicated in human diabetic complications, herein we evaluate diabetes in a unique mouse model that harbors a mitochondrial DNA from a divergent mouse species (the ‘xenomitochondrial mouse’), which has mild mitochondrial dysfunction and increased oxidative stress. We use the streptozotocin-induced diabetes model with insulin supplementation, with 20-weeks diabetes. We compare C57BL/6 mice and the ‘xenomitochondrial’ mouse, with measures of heart and kidney function, histology, and skin oxidative stress markers. Compared to C57BL/6 mice, the xenomitochondrial mouse has increased diabetic heart and kidney damage, with cardiac dysfunction, and increased cardiac and renal fibrosis. Our results show that mitochondrial oxidative stress consequent to divergent mtDNA can worsen diabetes complications. This has implications for novel therapeutics to counter diabetes complications, and for genetic studies of risk, as mtDNA genotypes may contribute to clinical outcomes.

Background. A disintegrin and metalloproteinase with thrombospondin motifs 1, Adamts-1, is important for the development and function of the kidney. Mice lacking this protein present with renal lesions comprising enlarged calyces, and reduced cortex and medulla layers. Our current findings are consistent with the defect occurring due to a developmental dysgenesis.Methods. We generated Adamts-1 null mice, and further investigated their kidney phenotype in a time course study ranging from E18.5 to 12 months of age. Immunohistochemistry was used to assess the localization of type IV collagen, TGF- and F4/80-positive macrophages in the kidneys of Adamts-1 null mice compared to wild-type control animals. The expression of Adamts-1 mRNA was determined in metanephric kidney explants by in situ hybridization.Results.Adamts-1 null mice have a gross kidney defect. At day 18.5 of gestation, the Adamts-1 null kidney has a normal appearance but at birth when the kidney begins to function, the defect becomes evident. During development of the kidney Adamts-1 expression was specifically detected in the developing loops of Henle, as well as in the proximal and distal convoluted tubules. Expression was not detected in the ureter, ureteric bud or its derivatives as had been previously suggested. At 6 months and 1 year of age, the Adamts-1 null mice displayed interstitial fibrosis in the cortical and medullary regions of the kidney. At 1 year of age, the Adamts-1 null mice displayed mild interstitial matrix expansion associated with increased collagen type IV expression, without apparent tubular dilatation, compared to wild-type animals. Immunohistochemical analysis demonstrated TGF- protein localized to infiltrating macrophages and glomeruli of Adamts-1 null mice.Conclusions. Adamts-1 is required for the normal development of the kidney. The defect observed in its absence results from a dysgenic malformation affecting the medulla that becomes apparent at birth, once the kidneys start to function. [ABSTRACT FROM AUTHOR]

Background. Hyperglycaemia is a causative factor in the pathogenesis of diabetic nephropathy, known to induce chemokines in the kidney. Macrophage inflammatory protein-3α (MIP-3α) is a CC chemokine that has been reported to attract memory T lymphocytes. Our previous microarray study showed significant increased level of MIP-3α in high glucose-induced transcriptional profile in renal proximal tubule cells. Transforming growth factor-β1 (TGF-β1) is a key regulator in inflammation and fibrosis in diabetes mellitus setting. Methods. This study aimed to determine the role of TGFβ1 in high glucose-induced MIP-3α expression. An in vitro model of human proximal tubular cells (HK-2 cells) and an in vivo model of the transgenic (mRen-2)27 diabetic rat, well characterized as a model of human diabetic nephropathy, were used. Small interfering RNA technology was used to silence TGF-β1 gene in HK-2 cells and subsequent experiments were performed to measure mRNA and protein levels of MIP-3α using real time reverse transcription–polymerase chain reaction (RT–PCR) and enzyme-linked immunosorbent assay (ELISA). Immunohistochemistry was used to measure the protein level of MIP-3α and CD3 a marker of T lymphocytes in the in vivo model. Results. MIP-3α mRNA and protein expression was increased in HK-2 cells by high glucose and TGF-β1. MIP-3α was up-regulated in the dilated tubules of diabetic rats compared with non-diabetic control animals and CD3 was found to be present around the dilated tubules expressing MIP-3α. This up-regulation was attenuated in the presence of an angiotensin-converting enzyme (ACE) inhibitor. MIP-3α expression significantly decreased in cells in which the TGF-β1 gene was silenced using small interfering RNA. Furthermore, exposure to high glucose did not induce MIP-3α expression in TGF-β1 gene silenced cells compared with wild-type cells. Conclusions. In summary, we have uniquely demonstrated that high glucose increases MIP-3α through a TGFβ1 dependent pathway, suggesting the centrality of TGF-β1 in both the inflammatory and previously demonstrated fibrotic responses in diabetic nephropathy. [ABSTRACT FROM AUTHOR]

Abstract Neovascular AMD (nAMD) leads to vision loss and is a leading cause of visual impairment in the industrialised world. Current treatments that target blood vessel growth have not been able to treat subretinal fibrosis and nAMD patients continue to lose vision. The molecular mechanisms involved in the development of fibrotic lesions in nAMD are not well understood. The aim of this study was to further understand subretinal fibrosis in the laser photocoagulation model of choroidal neovascularization (CNV) by studying the whole transcriptome of the RPE/choroid following CNV and the application of an anti-fibrotic following CNV. Seven days after laser induced CNV, RPE and choroid tissue was separated and underwent RNAseq. Differential expression analysis and pathway analysis revealed an over representation of immune signalling and fibrotic associated pathways in CNV compared to control RPE/choroid tissue. Comparisons between the mouse CNV model to human CNV revealed an overlap in upregulated expression for immune genes (Ccl2, Ccl8 and Cxcl9) and extracellular matrix remodeling genes (Comp, Lrcc15, Fndc1 and Thbs2). Comparisons between the CNV model and other fibrosis models showed an overlap of over 60% of genes upregulated in either lung or kidney mouse models of fibrosis. Treatment of CNV using a novel cinnamoyl anthranilate anti-fibrotic (OCX063) in the laser induced CNV model was selected as this class of drugs have previously been shown to target fibrosis. CNV lesion leakage and fibrosis was found to be reduced using OCX063 and gene expression of genes within the TGF-beta signalling pathway. Our findings show the presence of fibrosis gene expression pathways present in the laser induced CNV mouse model and that anti-fibrotic treatments offer the potential to reduce subretinal fibrosis in AMD.

Abstract Hypertension plays an important role in the development and progression of chronic kidney disease. Studies to date, with mineralocorticoid receptor antagonists (MRA), have demonstrated varying degrees of results in modifying the development of renal fibrosis. This study aimed to investigate whether treatment with a MRA commenced following the establishment of hypertension, a situation more accurately representing the clinical setting, modified the progression of renal fibrosis. Using male Cyp1a1Ren2 rats (n = 28), hypertension was established by addition of 0.167% indole‐3‐carbinol (w/w) to the rat chow, for 2 weeks prior to treatment. Rats were then divided into normotensive, hypertensive (H), or hypertensive with daily oral spironolactone treatment (H + SP) (human equivalent dose 50 mg/day). Physiological data and tissue were collected after 4 and 12 weeks for analysis. After 4 weeks, spironolactone had no demonstrable effect on systolic blood pressure (SBP), proteinuria, or macrophage infiltration in the renal cortex. However, glomerulosclerosis and renal cortical fibrosis were significantly decreased. Following 12 weeks of spironolactone treatment, SBP was lowered (not back to normotensive levels), proteinuria was reduced, and the progression of glomerulosclerosis and renal cortical fibrosis was significantly blunted. This was associated with a significant reduction in macrophage and myofibroblast infiltration, as well as CTGF and pSMAD2 expression. In summary, in a model of established hypertension, spironolactone significantly blunted the progression of renal fibrosis and glomerulosclerosis, and downregulated the renal inflammatory response, which was associated with reduced proteinuria, despite only a partial reduction in systolic blood pressure. This suggests a blood pressure independent effect of MRA on renal fibrosis.

The uremic toxin indoxyl sulfate (IS), elevated in chronic kidney disease (CKD), is known to contribute towards progressive cardiovascular disease. IS activates the aryl hydrocarbon receptor (AhR) mediating oxidative stress and endothelial dysfunction via activation of the CYP1A1 pathway. The present study examines AhR inhibition with the antagonist, CH223191, on IS-mediated impairment of vascular endothelial function and disruption of redox balance. The acute effects of IS on endothelium-dependent relaxation were assessed in aortic rings from Sprague Dawley rats exposed to the following conditions: (1) control; (2) IS (300 μM); (3) IS + CH223191 (1 μM); (4) IS + CH223191 (10 μM). Thereafter, tissues were assessed for changes in expression of redox markers. IS reduced the maximum level of endothelium-dependent relaxation (Rmax) by 42% (p < 0.001) compared to control, this was restored in the presence of increasing concentrations of CH223191 (p < 0.05). Rings exposed to IS increased expression of CYP1A1, nitro-tyrosine, NADPH oxidase 4 (NOX4), superoxide, and reduced eNOS expression (p < 0.05). CH223191 (10 μM) restored expression of these markers back to control levels (p < 0.05). These findings demonstrate the adverse impact of IS-mediated AhR activation on the vascular endothelium, where oxidative stress may play a critical role in inducing endothelial dysfunction in the vasculature of the heart and kidneys. AhR inhibition could provide an exciting novel therapy for CVD in the CKD setting.

Summary: Previous studies have shown that patients with Takotsubo syndrome (TS) have supranormal nitric oxide signaling, and post-mortem studies of TS heart samples revealed nitrosative stress. Therefore, we first showed in a female rat model that isoproterenol induces TS-like echocardiographic changes, evidence of nitrosative stress, and consequent activation of the energy-depleting enzyme poly(ADP-ribose) polymerase-1. We subsequently showed that pre-treatment with an inhibitor of poly(ADP-ribose) polymerase-1 ameliorated contractile abnormalities. These findings thus add to previous reports of aberrant β-adrenoceptor signaling (coupled with nitric oxide synthase activation) to elucidate mechanisms of impaired cardiac function in TS and point to potential methods of treatment. Key Words: myocardial inflammation, oxidative stress, poly(ADP-ribose) polymerase-1, Takotsubo cardiomyopathy

Background: The cannabinoid receptor type 2 (CB2) is reduced in podocytes of animals and humans with Type 2 Diabetes Mellitus (T2DM), with activation of CB2 ameliorating albuminuria in animals. As albuminuria also is due to proximal tubule dysfunction, the aim of this study is to investigate tubular expression of CB2 under diabetic conditions in addition to the cell signaling pathways that underlie these changes. Methods: We characterized total CB2 protein in diabetic animals and in Human Kidney 2 (HK2) cells exposed to elevated albumin and glucose, the levels of CB2 mRNA and protein. We also used latrunculin to determine if internalization of albumin was required to regulate CB2 levels. Finally, we characterized the levels of active and total AKT, ERK1/2 and p38 in response to albumin. Results: There were no changes to CB2 expression in kidney lysate from diabetic rats. In HK2 cells, expression of CB2 was unaltered following exposure to high glucose. High albumin treatment alone and in combination with high glucose, resulted in a significant reduction in CB2 receptor mRNA expression at 6 and 18 hours. CB2 protein expression was reduced at 6 and 24 hours, in high albumin and in combination with high glucose. Internalization of albumin was required to regulate CB2 levels, and inhibition of ERK1/2, did not rescue the loss of CB2 in response to albumin. Conclusion: We have demonstrated that internalization of albumin is required to reduce CB2 mRNA and protein expression in proximal tubules in vitro. Consequently, altered expression of CB2 in both the podocytes and tubules may contribute to the albuminuria observed in T2DM.

Diabetes mellitus significantly increases the risk of cardiovascular disease and heart failure in patients. Independent of hypertension and coronary artery disease, diabetes is associated with a specific cardiomyopathy, known as diabetic cardiomyopathy (DCM). Four decades of research in experimental animal models and advances in clinical imaging techniques suggest that DCM is a progressive disease, beginning early after the onset of type 1 and type 2 diabetes, ahead of left ventricular remodeling and overt diastolic dysfunction. Although the molecular pathogenesis of early DCM still remains largely unclear, activation of protein kinase C appears to be central in driving the oxidative stress dependent and independent pathways in the development of contractile dysfunction. Multiple subcellular alterations to the cardiomyocyte are now being highlighted as critical events in the early changes to the rate of force development, relaxation and stability under pathophysiological stresses. These changes include perturbed calcium handling, suppressed activity of aerobic energy producing enzymes, altered transcriptional and posttranslational modification of membrane and sarcomeric cytoskeletal proteins, reduced actin-myosin cross-bridge cycling and dynamics, and changed myofilament calcium sensitivity. In this review, we will present and discuss novel aspects of the molecular pathogenesis of early DCM, with a special focus on the sarcomeric contractile apparatus.