Inhibition of sodium glucose cotransporter 2 (SGLT2) has been reported as a new therapeutic strategy for treating diabetes. However, the effect of SGLT2 inhibitors on the kidney is unknown. In addition, whether SGLT2 inhibitors have an anti-inflammatory or antioxidative stress effect is still unclear. In this study, to resolve these issues, we evaluated the effects of the SGLT2 inhibitor, dapagliflozin, using a mouse model of type 2 diabetes and cultured proximal tubular epithelial (mProx24) cells. Male db/db mice were administered 0.1 or 1.0 mg/kg of dapagliflozin for 12 weeks. Body weight, blood pressure, blood glucose, hemoglobin A1c, albuminuria and creatinine clearance were measured. Mesangial matrix accumulation and interstitial fibrosis in the kidney and pancreatic β-cell mass were evaluated by histological analysis. Furthermore, gene expression of inflammatory mediators, such as osteopontin, monocyte chemoattractant protein-1 and transforming growth factor-β, was evaluated by quantitative reverse transcriptase-PCR. In addition, oxidative stress was evaluated by dihydroethidium and NADPH oxidase 4 staining. Administration of 0.1 or 1.0 mg/kg of dapagliflozin ameliorated hyperglycemia, β-cell damage and albuminuria in db/db mice. Serum creatinine, creatinine clearance and blood pressure were not affected by administration of dapagliflozin, but glomerular mesangial expansion and interstitial fibrosis were suppressed in a dose-dependent manner. Dapagliflozin treatment markedly decreased macrophage infiltration and the gene expression of inflammation and oxidative stress in the kidney of db/db mice. Moreover, dapagliflozin suppressed the high-glucose-induced gene expression of inflammatory cytokines and oxidative stress in cultured mProx24 cells. These data suggest that dapagliflozin ameliorates diabetic nephropathy by improving hyperglycemia along with inhibiting inflammation and oxidative stress.

Oxidative stress and inflammation play important roles in diabetic complications, including diabetic nephropathy. Metallothionein (MT) is induced in proximal tubular epithelial cells as an antioxidant in the diabetic kidney; however, the role of MT in renal function remains unclear. We therefore investigated whether MT deficiency accelerates diabetic nephropathy through oxidative stress and inflammation. Diabetes was induced by streptozotocin injection in MT-deficient (MT(-/-)) and MT(+/+) mice. Urinary albumin excretion, histological changes, markers for reactive oxygen species (ROS), and kidney inflammation were measured. Murine proximal tubular epithelial (mProx24) cells were used to further elucidate the role of MT under high-glucose conditions. Parameters of diabetic nephropathy and markers of ROS and inflammation were accelerated in diabetic MT(-/-) mice compared with diabetic MT(+/+) mice, despite equivalent levels of hyperglycemia. MT deficiency accelerated interstitial fibrosis and macrophage infiltration into the interstitium in the diabetic kidney. Electron microscopy revealed abnormal mitochondrial morphology in proximal tubular epithelial cells in diabetic MT(-/-) mice. In vitro studies demonstrated that knockdown of MT by small interfering RNA enhanced mitochondrial ROS generation and inflammation-related gene expression in mProx24 cells cultured under high-glucose conditions. The results of this study suggest that MT may play a key role in protecting the kidney against high glucose-induced ROS and subsequent inflammation in diabetic nephropathy.

Background/Aims: Telmisartan, an angiotensin II type 1 receptor blocker, is widely used to treat hypertension and kidney diseases, including diabetic nephropathy, because of its renoprotective effects. However, the mechanism by which telmisartan prevents proteinuria and renal dysfunction in diabetic nephropathy is still unclear. In this study, we examined the effects of telmisartan against diabetic nephropathy in db/db mice. Methods: Telmisartan was administered at a dose of 5 mg/kg/day for 3 weeks to db/db (diabetic) and db/m (control) mice. Urinary albumin excretion, renal histology, and the gene expression of oxidative stress and inflammatory markers in renal tissue were determined. To evaluate the effects of telmisartan on reactive oxygen species (ROS) production, superoxide was detected by dihydroethidium (DHE) staining in vivo and in vitro. Results: Telmisartan reduced albuminuria, mesangial matrix expansion, macrophage infiltration, and the expression of ROS markers (NADPH oxidase 4- and 8-hydroxydeoxyguanosine) and inflammatory cytokines (monocyte chemoattractant protein-1, osteopontin, and transforming growth factor-β) in the kidney. DHE staining showed that telmisartan decreased ROS generation in the kidney and in cultured mesangial and proximal tubular epithelial cells. Conclusions: Taken together, these findings indicate that telmisartan protects against diabetic nephropathy by reducing diabetes-induced oxidative stress.

Nuclear hormone receptors (NHRs) are transcription factors that regulate carbohydrate and lipid metabolism, immune responses, and inflammation. Although several NHRs, including peroxisome proliferator-activated receptor-γ (PPARγ) and PPARα, demonstrate a renoprotective effect in the context of diabetic nephropathy (DN), the expression and role of other NHRs in the kidney are still unrecognized. To investigate potential roles of NHRs in the biology of the kidney, we used quantitative real-time polymerase chain reaction to profile the expression of all 49 members of the mouse NHR superfamily in mouse kidney tissue (C57BL/6 and db/m), and cell lines of mesangial (MES13), podocyte (MPC), proximal tubular epithelial (mProx24) and collecting duct (mIMCD3) origins in both normal and high-glucose conditions. In C57BL/6 mouse kidney cells, hepatocyte nuclear factor 4α, chicken ovalbumin upstream promoter transcription factor II (COUP-TFII) and COUP-TFIII were highly expressed. During hyperglycemia, the expression of the NHR 4A subgroup including neuron-derived clone 77 (Nur77), nuclear receptor-related factor 1, and neuron-derived orphan receptor 1 significantly increased in diabetic C57BL/6 and db/db mice. In renal cell lines, PPARδ was highly expressed in mesangial and proximal tubular epithelial cells, while COUP-TFs were highly expressed in podocytes, proximal tubular epithelial cells, and collecting duct cells. High-glucose conditions increased the expression of Nur77 in mesangial and collecting duct cells, and liver x receptor α in podocytes. These data demonstrate NHR expression in mouse kidney cells and cultured renal cell lines and suggest potential therapeutic targets in the kidney for the treatment of DN.

Objective-Aberrant proliferation of smooth muscle cells (SMC) in response to injury induces pathological vascular remodeling during atherosclerosis and neointima formation. Telomerase is rate limiting for tissue renewal and cell replication; however, the physiological role of telomerase in vascular diseases remains to be determined. The goal of the present study was to determine whether telomerase reverse transcriptase (TERT) affects proliferative vascular remodeling and to define the molecular mechanism by which TERT supports SMC proliferation. Approach and Results-We first demonstrate high levels of TERT expression in replicating SMC of atherosclerotic and neointimal lesions. Using a model of guidewire-induced arterial injury, we demonstrate decreased neointima formation in TERT-deficient mice. Studies in SMC isolated from TERT-deficient and TERT overexpressing mice with normal telomere length established that TERT is necessary and sufficient for cell proliferation. TERT deficiency did not induce a senescent phenotype but resulted in G1 arrest albeit hyperphosphorylation of the retinoblastoma protein. This proliferative arrest was associated with stable silencing of the E2F1-dependent S-phase gene expression program and not reversed by ectopic overexpression of E2F1. Finally, chromatin immunoprecipitation and accessibility assays revealed that TERT is recruited to E2F1 target sites and promotes chromatin accessibility for E2F1 by facilitating the acquisition of permissive histone modifications. cultured cells, TERT is induced in both activated macrophages and proliferating SMC. 14,17 In SMC, the transient increase in TERT abundance in response to growth factor stimulation serves a mitogenic role because inhibition of TERT by oligonucleotide or pharmacological targeting inhibits SMC proliferation. 15,[17][18][19][20] Furthermore, acute telomerase downregulation using TERT antisense RNA decreases proliferation of SMC isolated from genetically hypertensive rats. 21 Although recent genetic experiments established that the mitogenic activity of TERT requires neither telomere extension nor the catalytic activity, 5-8 the molecular mechanisms by which TERT supports cellular proliferation remain elusive. Conclusions-TheseIn this study, we used a genetic model of TERT deficiency to investigate the transcriptional mechanisms underlying the mitogenic activity of TERT during proliferative vascular remodeling. Our studies reveal that TERT deletion prevents neointima formation and SMC proliferation by inducing a silenced chromatin environment at S phase gene promoters. Materials and MethodsMaterials and Methods are available in the online-only Data Supplement. Results TERT Is Expressed in Replicating Neointimal Smooth Muscle CellsTERT protein expression was first analyzed in neointimal SMC of human coronary artery atherosclerotic lesions from hearts explanted during cardiac transplantation. Although TERT expression is considered negligible in normal arteries, 13,14 we confirmed abundant TERT expression in the SMCrich neoint...