Darren A. Yuen

Long-Term Administration of the Histone Deacetylase Inhibitor Vorinostat Attenuates Renal Injury in Experimental Diabetes through an Endothelial Nitric Oxide Synthase-Dependent Mechanism

Epigenetic changes in gene expression play a role in the development of diabetic complications, including nephropathy. Histone deacetylases (HDACs) are a group of enzymes that exert epigenetic effects by altering the acetylation status of histone and nonhistone proteins. In the current study, we investigated the action of the clinically available HDAC inhibitor vorinostat in a mouse model of diabetic nephropathy, with the following aims: to define its effect on the progression of renal injury and to explore its mechanism of action by focusing on its role in regulating the expression of endothelial nitric oxide synthase (eNOS). Control and streptozotocin-diabetic wild-type and eNOS(-/-) mice were treated with vorinostat by daily oral dosing for 18 weeks. Without affecting either blood glucose concentration or blood pressure, vorinostat decreased albuminuria, mesangial collagen IV deposition, and oxidative-nitrosative stress in streptozotocin-wild-type mice. These attenuating effects were associated with a >50% reduction in eNOS expression in mouse kidneys and in cultured human umbilical vein endothelial cells. Vorinostat treatment had no effect on albuminuria, glomerular collagen IV concentration, or mesangiolysis in diabetic mice genetically deficient in eNOS. These observations illustrate the therapeutic efficacy of long-term HDAC inhibition in diabetic nephropathy and emphasize the importance of the interplay between eNOS activity and oxidative stress in mediating these effects.

eNOS Deficiency Predisposes Podocytes to Injury in Diabetes

Endothelial nitric oxide synthase (eNOS) deficiency may contribute to the pathogenesis of diabetic nephropathy in both experimental models and humans, but the underlying mechanism is not fully understood. Here, we studied two common sequelae of endothelial dysfunction in diabetes: glomerular capillary growth and effects on neighboring podocytes. Streptozotocin-induced diabetes increased glomerular capillary volume in both C57BL/6 and eNOS(-/-) mice. Inhibiting the vascular endothelial growth factor receptor attenuated albuminuria in diabetic C57BL/6 mice but not in diabetic eNOS(-/-) mice, even though it inhibited glomerular capillary enlargement in both. In eNOS(-/-) mice, an acute podocytopathy and heavy albuminuria occurred as early as 2 weeks after inducing diabetes, but treatment with either captopril or losartan prevented these effects. In vitro, serum derived from diabetic eNOS(-/-) mice augmented actin filament rearrangement in cultured podocytes. Furthermore, conditioned medium derived from eNOS(-/-) glomerular endothelial cells exposed to both high glucose and angiotensin II activated podocyte RhoA. Taken together, these results suggest that the combined effects of eNOS deficiency and hyperglycemia contribute to podocyte injury, highlighting the importance of communication between endothelial cells and podocytes in diabetes. Identifying mediators of this communication may lead to the future development of therapies targeting endothelial dysfunction in albuminuric individuals with diabetes.

Histone deacetylase inhibition attenuates diabetes-associated kidney growth: potential role for epigenetic modification of the epidermal growth factor receptor

Clinical trials and experimental studies have highlighted the importance of epigenetic processes in the development of diabetic complications. One of the earliest features of diabetic nephropathy is renal enlargement. The epidermal growth factor (EGF) has a pivotal role in the development of diabetic nephromegaly and transactivation of its receptor has been implicated in the pathogenesis of later-stage disease. As EGF signaling is altered by the acetylation status of histone proteins, we measured the effects of the histone deacetylase (HDAC) inhibitor, vorinostat, in mediating renal enlargement in diabetes focusing on the EGF-EGF receptor (EGFR) axis. In cultured proximal tubule (normal rat kidney) cells, vorinostat treatment reduced EGFR protein and mRNA, and attenuated cellular proliferation. Within 72 h of diabetes induction with streptozotocin, urinary EGF excretion was increased approximately threefold and was unaffected by vorinostat, even though the kidneys of vorinostat-treated diabetic rats had reduced tubular epithelial cell proliferation. Daily treatment of diabetic rats with vorinostat for 4 weeks blunted renal growth and glomerular hypertrophy. Thus, early renal changes in diabetes are amenable to epigenetic intervention. Attenuating effects of HDAC inhibition, although multifactorial, are likely to be mediated in part through downregulation of the EGFR.

The CXCR4/CXCR7/SDF-1 pathway contributes to the pathogenesis of Shiga toxin–associated hemolytic uremic syndrome in humans and mice

Hemolytic uremic syndrome (HUS) is a potentially life-threatening condition. It often occurs after gastrointestinal infection with E. coli O157:H7, which produces Shiga toxins (Stx) that cause hemolytic anemia, thrombocytopenia, and renal injury. Stx-mediated changes in endothelial phenotype have been linked to the pathogenesis of HUS. Here we report our studies investigating Stx-induced changes in gene expression and their contribution to the pathogenesis of HUS. Stx function by inactivating host ribosomes but can also alter gene expression at concentrations that minimally affect global protein synthesis. Gene expression profiling of human microvascular endothelium treated with Stx implicated a role for activation of CXCR4 and CXCR7 by their shared cognate chemokine ligand (stromal cell-derived factor-1 [SDF-1]) in Stx-mediated pathophysiology. The changes in gene expression required a catalytically active Stx A subunit and were mediated by enhanced transcription and mRNA stability. Stx also enhanced the association of CXCR4, CXCR7, and SDF1 mRNAs with ribosomes. In a mouse model of Stx-mediated pathology, we noted changes in plasma and tissue content of CXCR4, CXCR7, and SDF-1 after Stx exposure. Furthermore, inhibition of the CXCR4/SDF-1 interaction decreased endothelial activation and organ injury and improved animal survival. Finally, in children infected with E. coli O157:H7, plasma SDF-1 levels were elevated in individuals who progressed to HUS. Collectively, these data implicate the CXCR4/CXCR7/SDF-1 pathway in Stx-mediated pathogenesis and suggest novel therapeutic strategies for prevention and/or treatment of complications associated with E. coli O157:H7 infection.

YAP/TAZ Are Mechanoregulators of TGF-β-Smad Signaling and Renal Fibrogenesis

Like many organs, the kidney stiffens after injury, a process that is increasingly recognized as an important driver of fibrogenesis. Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) are related mechanosensory proteins that bind to Smad transcription factors, the canonical mediators of profibrotic TGF-β responses. Here, we investigated the role of YAP/TAZ in the matrix stiffness dependence of fibroblast responses to TGF-β In contrast to growth on a stiff surface, fibroblast growth on a soft matrix led to YAP/TAZ sequestration in the cytosol and impaired TGF-β-induced Smad2/3 nuclear accumulation and transcriptional activity. YAP knockdown or treatment with verteporfin, a drug that was recently identified as a potent YAP inhibitor, elicited similar changes. Furthermore, verteporfin reduced YAP/TAZ levels and decreased the total cellular levels of Smad2/3 after TGF-β stimulation. Verteporfin treatment of mice subjected to unilateral ureteral obstruction similarly reduced YAP/TAZ levels and nuclear Smad accumulation in the kidney, and attenuated renal fibrosis. Our data suggest that organ stiffening cooperates with TGF-β to induce fibrosis in a YAP/TAZ- and Smad2/3-dependent manner. Interference with this YAP/TAZ and TGF-β/Smad crosstalk likely underlies the antifibrotic activity of verteporfin. Finally, through repurposing of a clinically used drug, we illustrate the therapeutic potential of a novel mechanointerference strategy that blocks TGF-β signaling and renal fibrogenesis.

DPP-4 Inhibition Attenuates Cardiac Dysfunction and Adverse Remodeling Following Myocardial Infarction in Rats with Experimental Diabetes

AIMS Following myocardial infarction (MI), individuals with diabetes have a two-fold increase in the risk of heart failure, due in part to excessive loss of cardiac microvasculature. Endothelial integrity and restitution are mediated in part by stromal cell-derived factor-1α (SDF-1α), a chemokine that is elaborated by ischemic tissue but rapidly degraded by dipeptidyl peptidase-4 (DPP-4). Accordingly, we hypothesized that inhibiting this enzyme may confer benefit following myocardial infarction in the diabetic setting beyond its effect on glycemia. METHODS AND RESULTS Fischer F344 rats with streptozotocin (STZ)-diabetes were randomized to receive vehicle or the DPP-4 inhibitor, sitagliptin (300 mg/kg/day). Two weeks later, animals underwent experimental MI, induced by ligation of the left anterior descending coronary artery. Cardiac function was assessed by conductance catheterization and echocardiography along with cardiac structure 4 weeks post-MI. Following MI, untreated diabetic rats developed both systolic and diastolic cardiac dysfunction, in association with endothelial cell loss, fibrosis, and myocyte hypertrophy. Without affecting plasma glucose, sitagliptin treatment led to an improvement in passive left ventricular compliance, increased endothelial cell density, reduced myocyte hypertrophy, and a reduction in the abundance of collagen 1 (all P < 0.05). Systolic function was unchanged. CONCLUSIONS This study shows that DPP-4 inhibition attenuates several, but not all, aspects of cardiac dysfunction and adverse remodeling in the post-MI setting.

Culture-modified bone marrow cells attenuate cardiac and renal injury in a chronic kidney disease rat model via a novel antifibrotic mechanism.

Background 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). Methodology/Principal Findings In vitro, CMC-conditioned medium, but not SC-conditioned medium, inhibited fibroblast collagen production and cell signalling in response to transforming growth factor-ß. 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 106 SCs had no effect, 106 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<0.05 for both). Similarly, 106 CMCs reduced cardiac fibrosis compared to saline (% area stained with picrosirius red: 3.2±0.3 v 5.1±0.4, p<0.05), whereas 106 SCs had no effect. Structural changes induced by CMC therapy were accompanied by improved function, as reflected by reductions in plasma creatinine (58±3 v 81±11 µmol/L), urinary protein excretion (9×/÷1 v 64×/÷1 mg/day), and diastolic cardiac stiffness (left ventricular end-diastolic pressure-volume relationship: 0.030±0.003 v 0.058±0.011 mm Hg/µL, p<0.05 for all). Despite substantial improvements in structure and function, only rare CMCs were present in the kidney and heart, whereas abundant CMCs were detected in the liver and spleen. Conclusions/Significance Together, these findings provide the first evidence suggesting that CMCs, but not SCs, exert a protective action in cardio-renal disease and that these effects may be mediated by the secretion of diffusible anti-fibrotic factor(s).

Fluorescent Microangiography Is a Novel and Widely Applicable Technique for Delineating the Renal Microvasculature

Rarefaction of the renal microvasculature correlates with declining kidney function. However, current technologies commonly used for its evaluation are limited by their reliance on endothelial cell antigen expression and assessment in two dimensions. We set out to establish a widely applicable and unbiased optical sectioning method to enable three dimensional imaging and reconstruction of the renal microvessels based on their luminal filling. The kidneys of subtotally nephrectomized (SNx) rats and their sham-operated counterparts were subjected to either routine two-dimensional immunohistochemistry or the novel technique of fluorescent microangiography (FMA). The latter was achieved by perfusion of the kidney with an agarose suspension of fluorescent polystyrene microspheres followed by optical sectioning of 200 µm thick cross-sections using a confocal microscope. The fluorescent microangiography method enabled the three-dimensional reconstruction of virtual microvascular casts and confirmed a reduction in both glomerular and peritubular capillary density in the kidneys of SNx rats, despite an overall increase in glomerular volume. FMA is an uncomplicated technique for evaluating the renal microvasculature that circumvents many of the limitations imposed by conventional analysis of two-dimensional tissue sections.

Slit2 Prevents Neutrophil Recruitment and Renal Ischemia-Reperfusion Injury

Neutrophils recruited to the postischemic kidney contribute to the pathogenesis of ischemia-reperfusion injury (IRI), which is the most common cause of renal failure among hospitalized patients. The Slit family of secreted proteins inhibits chemotaxis of leukocytes by preventing activation of Rho-family GTPases, suggesting that members of this family might modulate the recruitment of neutrophils and the resulting IRI. Here, in static and microfluidic shear assays, Slit2 inhibited multiple steps required for the infiltration of neutrophils into tissue. Specifically, Slit2 blocked the capture and firm adhesion of human neutrophils to inflamed vascular endothelial barriers as well as their subsequent transmigration. To examine whether these observations were relevant to renal IRI, we administered Slit2 to mice before bilateral clamping of the renal pedicles. Assessed at 18 hours after reperfusion, Slit2 significantly inhibited renal tubular necrosis, neutrophil and macrophage infiltration, and rise in plasma creatinine. In vitro, Slit2 did not impair the protective functions of neutrophils, including phagocytosis and superoxide production, and did not inhibit neutrophils from killing the extracellular pathogen Staphylococcus aureus. In vivo, administration of Slit2 did not attenuate neutrophil recruitment or bacterial clearance in mice with ascending Escherichia coli urinary tract infections and did not increase the bacterial load in the livers of mice infected with the intracellular pathogen Listeria monocytogenes. Collectively, these results suggest that Slit2 may hold promise as a strategy to combat renal IRI without compromising the protective innate immune response.

Early-Outgrowth Bone Marrow Cells Attenuate Renal Injury and Dysfunction Via an Antioxidant Effect in a Mouse Model of Type 2 Diabetes

Cell therapy has been extensively investigated in heart disease but less so in the kidney. We considered whether cell therapy also might be useful in diabetic kidney disease. Cognizant of the likely need for autologous cell therapy in humans, we sought to assess the efficacy of donor cells derived from both healthy and diabetic animals. Eight-week-old db/db mice were randomized to receive a single intravenous injection of PBS or 0.5 × 106 early-outgrowth cells (EOCs) from db/m or db/db mice. Effects were assessed 4 weeks after cell infusion. Untreated db/db mice developed mesangial matrix expansion and tubular epithelial cell apoptosis in association with increased reactive oxygen species (ROS) and overexpression of thioredoxin interacting protein (TxnIP). Without affecting blood glucose or blood pressure, EOCs not only attenuated mesangial and peritubular matrix expansion, as well as tubular apoptosis, but also diminished ROS and TxnIP overexpression in the kidney of db/db mice. EOCs derived from both diabetic db/db and nondiabetic db/m mice were equally effective in ameliorating kidney injury and oxidative stress. The similarly beneficial effects of cells from healthy and diabetic donors highlight the potential of autologous cell therapy in the related clinical setting.