Danegaptide Prevents TGFβ1-Induced Damage in Human Proximal Tubule Epithelial Cells of the Kidney

Abstract

Chronic kidney disease (CKD) is a global health problem associated with a number of comorbidities. Recent evidence implicates increased hemichannel-mediated release of adenosine triphosphate (ATP) in the progression of tubulointerstitial fibrosis, the main underlying pathology of CKD. Here, we evaluate the effect of danegaptide on blocking hemichannel-mediated changes in the expression and function of proteins associated with disease progression in tubular epithelial kidney cells. Primary human proximal tubule epithelial cells (hPTECs) were treated with the beta1 isoform of the pro-fibrotic cytokine transforming growth factor (TGFβ1) ± danegaptide. qRT-PCR and immunoblotting confirmed mRNA and protein expression, whilst a cytokine antibody array assessed the expression/secretion of proinflammatory and profibrotic cytokines. Carboxyfluorescein dye uptake and ATP biosensing measured hemichannel activity and ATP release, whilst transepithelial electrical resistance was used to assess paracellular permeability. Danegaptide negated carboxyfluorescein dye uptake and ATP release and protected against protein changes associated with tubular injury. Blocking Cx43-mediated ATP release was paralleled by partial restoration of the expression of cell cycle inhibitors, adherens and tight junction proteins and decreased paracellular permeability. Furthermore, danegaptide inhibited TGFβ1-induced changes in the expression and secretion of key adipokines, cytokines, chemokines, growth factors and interleukins. The data suggest that as a gap junction modulator and hemichannel blocker, danegaptide has potential in the future treatment of CKD.

Connexin 43: A Target for the Treatment of Inflammation in Secondary Complications of the Kidney and Eye in Diabetes

“…Although a glomerular disease in origin, advanced stages of nephropathy are characterised by severe tubule interstitial inflammation and fibrosis [ 150 ]. Work within our laboratories links altered Cx43 expression to tubule injury in both in vitro [ 108 , 135 , 151 , 152 ] and in vivo [ 108 ] models of disease [ 153 ]. Initial observations identified an approximate 5-fold increase in Cx43 expression in biopsy material from individuals with diabetic nephropathy compared to healthy control [ 151 ], whilst paired-patch electrophysiology and ATP biosensing suggested that this increased expression was paralleled by diminished gap-junction intercellular coupling (GJIC) and increased hemichannel mediated ATP release [ 151 ].…”

“…Building on our published observations with Peptide 5, we recently assessed the efficacy of Danegaptide in conferring protection in an in vitro model of tubular injury. A Cx43 gap junction modifier [ 133 , 134 ], we reported that Danegaptide was also able to block hemichannel mediated dye uptake, ATP release and consequently TGFβ1 induced changes in markers of tubular injury e.g., E-Cadherin and N-Cadherin and fibrosis e.g., collagen-I, collagen-IV and fibronectin in human primary tubule epithelial cells (hPTECs) [ 135 ]. Furthermore, based on evidence that the Cx43 +/− UUO mouse presents with decreased fibroblast activation and diminished macrophage infiltration as compared to wild type UUO control [ 168 ], we employed proteome profiler arrays to screen for the expression profile of 125 inflammatory cytokines in TGFβ1 treated human primary proximal tubule cells in the presence/absence of Danegaptide [ 135 ].…”

“…A Cx43 gap junction modifier [ 133 , 134 ], we reported that Danegaptide was also able to block hemichannel mediated dye uptake, ATP release and consequently TGFβ1 induced changes in markers of tubular injury e.g., E-Cadherin and N-Cadherin and fibrosis e.g., collagen-I, collagen-IV and fibronectin in human primary tubule epithelial cells (hPTECs) [ 135 ]. Furthermore, based on evidence that the Cx43 +/− UUO mouse presents with decreased fibroblast activation and diminished macrophage infiltration as compared to wild type UUO control [ 168 ], we employed proteome profiler arrays to screen for the expression profile of 125 inflammatory cytokines in TGFβ1 treated human primary proximal tubule cells in the presence/absence of Danegaptide [ 135 ]. Soluble chemokines, adhesion molecules and growth factors recruit and activate infiltrating immune cells and resident fibroblasts to mediate inflammation and fibrosis in the diabetic kidney [ 135 ].…”

Mechanisms of Connexin Regulating Peptides

“…Despite their failure to decrease phenotypic disease burden, AAP10, rotagaptide, and danegaptide do alter the pattern of Cx43 expression, phosphorylation, GJ signaling, and hemichannel signaling [20,[72][73][74]. Studies have demonstrated that these peptides increase both Cx43 and Cx40 GJ signaling [72,75]. Unlike the peptides to be discussed later in this review, the AAP10 derivative peptides do not contain sequence homology with connexin proteins.…”

Mechanisms of Connexin Mimetic Peptides

“…Despite their failure to decrease phenotypic disease burden, AAP10, rotagaptide, and danegaptide do alter the pattern of Cx43 expression, phosphorylation, GJ signaling, and hemichannel signaling [17,[69][70][71]. Studies have demonstrated that these peptides increase both Cx43 and Cx40 GJ signaling [69,72]. Unlike the peptides to be discussed later in this review, the AAP10 derivative peptides do not contain sequence homology with connexin proteins.…”