tram-34 and Diabetic-Nephropathies

Despite optimal control of hyperglycaemia, hypertension, and dyslipidaemia, the number of patients with diabetic nephropathy (DN) continues to grow. Strategies to target various signaling pathways to prevent DN have been intensively investigated in animal models and many have been proved to be promising. However, targeting these pathways once kidney disease is established, remain unsatisfactory. The clinical scenario is that patients with diabetes mellitus often present with established kidney damage and need effective treatments to repair and reverse the kidney damage. In this studies, eNOS-/- mice were administered with streptozotocin to induce diabetes. At 24 weeks, at which time we have previously demonstrated albuminuria and pathological changes of diabetic nephropathy, mice were randomised to receive TRAM34 subcutaneously, a highly selective inhibitor of potassium channel KCa3.1 or DMSO (vehicle) for a further 14 weeks. Albuminuria was assessed, inflammatory markers (CD68, F4/80) and extracellular matrix deposition (type I collagen and fibronectin) in the kidneys were examined. The results clearly demonstrate that TRAM34 reduced albuminuria, decreased inflammatory markers and reversed extracellular matrix deposition in kidneys via inhibition of the TGF-β1 signaling pathway. These results indicate that KCa3.1 blockade effectively reverses established diabetic nephropathy in this rodent model and provides a basis for progressing to human studies.

Topics: Albuminuria; Animals; Collagen Type I; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Extracellular Matrix; Fibronectins; Heart; Hypoglycemic Agents; Intermediate-Conductance Calcium-Activated Potassium Channels; Kidney; Liver; Male; Mice, Knockout; Myocardium; Nitric Oxide Synthase Type III; Potassium Channel Blockers; Pyrazoles; Random Allocation; Spleen

Fibroblast activation plays a critical role in diabetic nephropathy (DN). The Ca2+-activated K+ channel KCa3.1 mediates cellular proliferation of many cell types including fibroblasts. KCa3.1 has been reported to be a potential molecular target for pharmacological intervention in a diverse array of clinical conditions. However, the role of KCa3.1 in the activation of myofibroblasts in DN is unknown. These studies assessed the effect of KCa3.1 blockade on renal injury in experimental diabetes.. As TGF-β1 plays a central role in the activation of fibroblasts to myofibroblasts in renal interstitial fibrosis, human primary renal interstitial fibroblasts were incubated with TGF-β1+/- the selective inhibitor of KCa3.1, TRAM34, for 48 h. Two streptozotocin-induced diabetic mouse models were used in this study: wild-type KCa3.1+/+ and KCa3.1-/- mice, and secondly eNOS-/- mice treated with or without a selective inhibitor of KCa3.1 (TRAM34). Then, markers of fibroblast activation and fibrosis were determined.. Blockade of KCa3.1 inhibited the upregulation of type I collagen, fibronectin, α-smooth muscle actin, vimentin and fibroblast-specific protein-1 in renal fibroblasts exposed to TGF-β1 and in kidneys from diabetic mice. TRAM34 reduced TGF-β1-induced phosphorylation of Smad2/3 and ERK1/2 but not P38 and JNK MAPK in interstitial fibroblasts.. These results suggest that blockade of KCa3.1 attenuates diabetic renal interstitial fibrogenesis through inhibiting activation of fibroblasts and phosphorylation of Smad2/3 and ERK1/2. Therefore, therapeutic interventions to prevent or ameliorate DN through targeted inhibition of KCa3.1 deserve further consideration.

Topics: Animals; Biopsy; Blotting, Western; Cell Proliferation; Cells, Cultured; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Fibroblasts; Fibrosis; Gene Expression Regulation; Humans; Immunohistochemistry; Intermediate-Conductance Calcium-Activated Potassium Channels; Kidney Cortex; Male; Mice; Mice, Inbred C57BL; Pyrazoles; Real-Time Polymerase Chain Reaction; RNA; Signal Transduction

The Ca(2+)-activated K(+) channel KCa3.1 mediates cellular signaling processes associated with dysfunction of vasculature. However, the role of KCa3.1 in diabetic nephropathy is unknown. We sought to assess whether KCa3.1 mediates the development of renal fibrosis in two animal models of diabetic nephropathy. Wild-type and KCa3.1(-/-) mice, and secondly eNOS(-/-) mice, had diabetes induced with streptozotocin and then were treated with/without a selective inhibitor of KCa3.1 (TRAM34). Our results show that the albumin-to-creatinine ratio significantly decreased in diabetic KCa3.1(-/-) mice compared with diabetic wild-type mice and in diabetic eNOS(-/-) mice treated with TRAM34 compared with diabetic mice. The expression of monocyte chemoattractant protein-1 (MCP-1), intercellular adhesion molecule 1 (ICAM1), F4/80, plasminogen activator inhibitor type 1 (PAI-1), and type III and IV collagen significantly decreased (P < 0.01) in kidneys of diabetic KCa3.1(-/-) mice compared with diabetic wild-type mice. Similarly, TRAM34 reduced the expression of the inflammatory and fibrotic markers described above in diabetic eNOS(-/-) mice. Furthermore, blocking the KCa3.1 channel in both animal models led to a reduction of transforming growth factor-β1 (TGF-β1) and TGF-β1 type II receptor (TβRII) and phosphorylation of Smad2/3. Our results provide evidence that KCa3.1 mediates renal fibrosis in diabetic nephropathy through the TGF-β1/Smad signaling pathway. Blockade of KCa3.1 may be a novel target for therapeutic intervention in patients with diabetic nephropathy.

Topics: Animals; Chemokine CCL2; Collagen Type IV; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Fibrosis; Humans; Intermediate-Conductance Calcium-Activated Potassium Channels; Kidney; Mice; Mice, Knockout; Plasminogen Activator Inhibitor 1; Pyrazoles; Receptors, Transforming Growth Factor beta; Signal Transduction; Smad Proteins; Transforming Growth Factor beta1

Topics: Animals; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Humans; Intermediate-Conductance Calcium-Activated Potassium Channels; Pyrazoles; Smad Proteins; Transforming Growth Factor beta1