title: High glucose induces CCL20 in proximal tubular cells via activation of the KCa3.1 channel.
abstract: BACKGROUND
  Inflammation plays a key role in the development and progression of diabetic nephropathy (DN). KCa3.1, a calcium activated potassium channel protein, is associated with vascular inflammation, atherogenesis, and proliferation of endothelial cells, macrophages, and fibroblasts. We have previously demonstrated that the KCa3.1 channel is activated by TGF-β1 and blockade of KCa3.1 ameliorates renal fibrotic responses in DN through inhibition of the TGF-β1 pathway. The present study aimed to identify the role of KCa3.1 in the inflammatory responses inherent in DN.
  
  
  METHODS
  Human proximal tubular cells (HK2 cells) were exposed to high glucose (HG) in the presence or absence of the KCa3.1 inhibitor TRAM34 for 6 days. The proinflammatory cytokine chemokine (C-C motif) ligand 20 (CCL20) expression was examined by real-time PCR and enzyme-linked immunosorbent assay (ELISA). The activity of nuclear factor-κB (NF-κB) was measured by nuclear extraction and electrophoretic mobility shift assay (EMSA). In vivo, the expression of CCL20, the activity of NF-κB and macrophage infiltration (CD68 positive cells) were examined by real-time PCR and/or immunohistochemistry staining in kidneys from diabetic or KCa3.1-/- mice, and in eNOS-/- diabetic mice treated with the KCa3.1 channel inhibitor TRAM34.
  
  
  RESULTS
  In vitro data showed that TRAM34 inhibited CCL20 expression and NF-κB activation induced by HG in HK2 cells. Both mRNA and protein levels of CCL20 significantly decreased in kidneys of diabetic KCa3.1-/- mice compared to diabetic wild type mice. Similarly, TRAM34 reduced CCL20 expression and NF-κB activation in diabetic eNOS-/- mice compared to diabetic controls. Blocking the KCa3.1 channel in both animal models led to a reduction in phosphorylated NF-κB.
  
  
  CONCLUSIONS
  Overexpression of CCL20 in human proximal tubular cells is inhibited by blockade of KCa3.1 under diabetic conditions through inhibition of the NF-κB pathway.
authors.affiliation: Kolling Institute of Medical Research, Royal North Shore Hospital, Sydney Medical School, University of Sydney, Sydney, NSW, Australia; Xiamen Center of Clinical Laboratory, Xiamen Zhongshan Hospital, Medical College of Xiamen University, Xiamen, China.
authors.forename: Chunling
authors.initials: C
authors.lastname: Huang
authors.affiliation: Kolling Institute of Medical Research, Royal North Shore Hospital, Sydney Medical School, University of Sydney, Sydney, NSW, Australia.
authors.forename: Carol A
authors.initials: CA
authors.lastname: Pollock
authors.affiliation: Kolling Institute of Medical Research, Royal North Shore Hospital, Sydney Medical School, University of Sydney, Sydney, NSW, Australia.
authors.forename: Xin-Ming
authors.initials: XM
authors.lastname: Chen
mesh_terms: D000818:Animals; D002460:Cell Line; D054418:Chemokine CCL20 / Q000378:metabolism*; D003921:Diabetes Mellitus, Experimental / Q000378:metabolism / Q000473:pathology; D005947:Glucose / Q000494:pharmacology*; D006801:Humans; D051660:Intermediate-Conductance Calcium-Activated Potassium Channels / Q000037:antagonists & inhibitors / Q000172:deficiency / Q000378:metabolism*; D015640:Ion Channel Gating / Q000187:drug effects*; D007687:Kidney Tubules, Proximal / Q000378:metabolism* / Q000473:pathology; D008264:Macrophages / Q000187:drug effects / Q000378:metabolism; D008297:Male; D008810:Mice, Inbred C57BL; D016328:NF-kappa B / Q000378:metabolism; D011720:Pyrazoles / Q000494:pharmacology; D015854:Up-Regulation / Q000187:drug effects
chemical_list: C108355:CCL20 protein, human; C493447:CCL20 protein, mouse; D054418:Chemokine CCL20; D051660:Intermediate-Conductance Calcium-Activated Potassium Channels; C496923:KCNN4 protein, human; C496925:Kcnn4 protein, mouse; D016328:NF-kappa B; D011720:Pyrazoles; C411671:TRAM 34; D005947:Glucose
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