Glomerulosclerosis and diabetic nephropathy are attributable to high glucose induction of mesangial cell apoptosis. Whereas Wnt signaling has been found to regulate renal morphogenesis and pathogenesis, the biologic role of Wnt/␤-catenin signaling in controlling high glucose-induced mesangial cell apoptosis is not well defined. Herein is reported that Wnt/␤-catenin signaling is required for protecting glomerular mesangial cells from high glucose-mediated cell apoptosis. High glucose downregulated Wnt4 and Wnt5a expression and the subsequent nuclear translocation of ␤-catenin, whereas it increased glycogen synthase kinase-3␤ (GSK-3␤) and caspase-3 activities and apoptosis of glomerular mesangial cells. Suppression of GSK-3␤ activation or increase in nuclear ␤-catenin by transfection of Wnt4 or Wnt5a or stable ␤-catenin (S33Y) reversed Akt activation and reduced the high glucose-mediated caspase-3 cleavage and cell apoptosis. Pharmacologic inhibition of GSK-3␤ by recombinant Wnt5a or bromoindirubin-3-oxime or LiCl increased Akt phosphorylation and ␤-catenin translocation and abrogated high glucose-mediated proapoptotic activities. Exogenous bromoindirubin-3-oxime treatment reduced phosphoSer 9 -GSK-3␤ and ␤-catenin expression and apoptosis of cells adjacent to glomeruli in diabetic kidneys and attenuated urinary protein secretion in diabetic rats. Taken together, mesangial cells responded to high glucose by impairing that canonical Wnt pathway to increase proapoptotic activities. Sustaining Wnt/␤-catenin signaling is beneficial for promoting survival of mesangial cells that are exposed to high glucose stress.

Wnt/␤-catenin signaling mediates renal fibrosis in several model systems including diabetic nephropathy. Dickkopf-1 (DKK-1) is an endogenous inhibitor of Wnt/␤-catenin signaling, but whether DKK-1 modulates diabetic nephropathy is unknown. Here, we studied whether DKK-1 participates in high glucose (HG)-induced expression of profibrotic factors and renal damage. In vitro, HG increased expression of DKK1, receptor Kremen-2, TGF-␤1, and fibronectin in mesangial cells. Loss and gain of DKK1 function modulated HG-mediated c-Jun, TGF-␤1, and fibronectin expression. DKK1 mediated HG-induced phosphorylation of Ser45-␤-catenin and reduction of nuclear ␤-catenin levels, but not phosphorylation of ERK kinase. Wnt3a protein and the ␤-catenin (⌬45) mutation increased nuclear ␤-catenin but abrogated HG-induced DKK1 and fibronectin expression. Exogenous DKK1 antisense oligonucleotide attenuated the increase in both serum DKK1 and urinary protein excretion in streptozotocin-induced diabetic rats. Knocking down DKK1 inhibited mesangial expression of TGF-␤1 and fibronectin and reduced both the glomerular volume and deposition of mesangial matrix in diabetic kidneys. Taken together, DKK1 mediates HG-induced destabilization of ␤-catenin and matrix accumulation in mesangial cells. Knocking down DKK1 prevents diabetes-induced renal dysfunction and microstructure deterioration, suggesting that inhibition of DKK1offers therapeutic potential for diabetic nephropathy. 21: 124 -135, 201021: 124 -135, . doi: 10.1681 Diabetes-mediated renal fibrosis is one of the leading causes of ESRD. High glucose (HG) concentration increases remodeling of glomerular microarchitecture by promoting accumulation of extracellular matrix (ECM) 1 and expression of fibrotic factor in mesangial cells, 2 which leads to renal dysfunction and subsequent diabetic renal injury. 3,4 Induction of fibrotic matrix deposition in mesangial cells by TGF-␤1 is an important pathogenic reaction in diabetic renal injury. 5 Wnt proteins interact with receptor Frizzled and co-receptor LDL receptor-related protein 5 (LRP5) and stabilize downstream transcription regulator ␤-catenin by inhibiting ␤-catenin phosphorylation toward proteasome degradation, 6 which reportedly participates in nephrogenesis and renal disorders. Modulation of Wnt protein secretion, ␤-catenin stabilization, and glycogen synthesis kinase-3␤ activa- J Am Soc Nephrol

Although previous studies have demonstrated that diabetic nephropathy is attributable to early extracellular matrix accumulation in glomerular mesangial cells, the molecular mechanism by which high glucose induces matrix protein deposition remains not fully elucidated. Rat mesangial cells pretreated with or without inhibitors were cultured in high-glucose or advanced glycation end product (AGE) conditions. Streptozotocin-induced diabetic rats were given superoxide dismutase (SOD)-conjugated propylene glycol to scavenge superoxide. Transforming growth factor (TGF)-beta1, fibronectin expression, Ras, ERK, p38, and c-Jun activation of glomerular mesangial cells or urinary albumin secretion were assessed. Superoxide, not nitric oxide or hydrogen peroxide, mediated high glucose- and AGE-induced TGF-beta1 and fibronectin expression. Pretreatment with diphenyliodonium, not allopurinol or rotenone, reduced high-glucose and AGE augmentation of superoxide synthesis and fibronection expression. High glucose and AGEs rapidly enhanced Ras activation and progressively increased cytosolic ERK and nuclear c-Jun activation. Inhibiting Ras by manumycin A reduced the stimulatory effects of high glucose and AGEs on superoxide and fibronectin expression. SOD or PD98059 pretreatment reduced high-glucose and AGE promotion of ERK and c-Jun activation. Exogenous SOD treatment in diabetic rats significantly attenuated diabetes induction of superoxide, urinary albumin excretion, 8-hydroxy-2'-deoxyguanosine, TGF-beta1, and fibronectin immunoreactivities in renal glomerular mesangial cells. Ras induction of superoxide activated ERK-dependent fibrosis-stimulatory factor and extracellular matrix gene transcription of mesangial cells. Reduction of oxidative stress by scavenging superoxide may provide an alternative strategy for controlling diabetes-induced early renal injury.

Intense mesangial cell apoptosis contributes to the pathogenesis of diabetic nephropathy. Although reactive oxygen radicals and Wnt signaling components are potent regulators that modulate renal tissue remodeling and morphogenesis, cross-talk between oxidative stress and Wnt/beta-catenin signaling in controlling high-glucose-impaired mesangial cell survival and renal function have not been tested. In this study, high glucose induced Ras and Rac1 activation, superoxide burst, and Wnt5a/beta-catenin destabilization and subsequently promoted caspase-3 and poly (ADP-ribose) polymerase cleavage and apoptosis in mesangial cell cultures. The pharmacological and genetic suppression of superoxide synthesis by superoxide dismutase and diphenyloniodium, dominant-negative Ras (S17N), and dominant-negative Rac1 (T17N) abrogated high-glucose-induced glycogen synthase kinase (GSK-3beta) activation and caspase-3 and poly (ADP-ribose) polymerase degradation. Inactivation of Ras and Racl also reversed Wnt/beta-catenin expression and survival of mesangial cells. Stabilization of beta-catenin by the transfection of stable beta-catenin (Delta45) and kinase-inactive GSK-3beta attenuated high-glucose-mediated mesangial cell apoptosis. Exogenous superoxide dismutase administration attenuated urinary protein secretion in diabetic rats and abrogated diabetes-mediated reactive oxygen radical synthesis in renal glomeruli. Immunohistological observation revealed that superoxide dismutase treatment abrogated diabetes-induced caspase-3 cleavage and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end-labeling (TUNEL) and increased Wnt5a/beta-catenin expression in renal glomeruli. Taken together, high glucose induced oxidative stress and apoptosis in mesangial cells. The Ras and Rac1 regulation of superoxide appeared to raise apoptotic activity by activating GSK-3beta and inhibiting Wnt5a/beta-catenin signaling. Controlling oxidative stress and Wnt/beta-catenin signaling has potential for protecting renal tissue against the deleterious effect of high glucose.

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