S REDZEPAGIC1, C POLLOCK1
1Renal Research Group, Kolling Institute, Royal North Shore Hospital, University of Sydney , Sydney, Australia
Aim: To assess if knockdown of primary cilia gene C2cd3 reduces TGF-β1-induced upregulation of kidney fibrosis and inflammation markers via reduced activation of Hedgehog signalling (HHS) pathway.
Background: We previously found a novel long-non-coding RNA Gm10603 is upregulated in DKD and by TGF-β1 in kidney PTCs, and identified C2cd3 and Ucp2 as its flanking genes. C2cd3 is essential in primary cilia HHS-pathway. We demonstrated a potential role of C2CD3 by upregulation in mouse and human models of DKD and TGF-β1 in kidney PTCs.
Methods: Primary cultures of mouse kidney PTCs and human kidney PTCs HK-2 were transfected with control and C2CD3-sequence-specific LNA-GapmeRs (Qiagen). Mouse and human kidney PTCs were exposed to TGF-β1 5ng/ml or 2ng/ml for 48hours and mRNA extracted. mRNA expression of C2CD3 and downstream fibrotic, inflammatory and HHS-pathway markers were assessed.
Results: Knockdown of C2cd3 by C2cd3-sequence-specific LNA-GapmeRs reduced TGF-β1-induced upregulation of C2cd3 (P<0.01), Collagen I (P<0.01), Fibronectin (P<0.05) and IL6 (P<0.05) in mouse kidney PTCs. In HK-2 PTCs, knockdown of C2CD3 by C2CD3-sequence-specific LNA-GapmeRs similarly reduced TGF-β1-induced upregulation of fibrotic and inflammatory markers Collagen IV (P<0.05), Fibronectin (P<0.01), PAI1 (P<0.01) and IL6 (P<0.001). TGF-β1 upregulated components of the HHS-pathway including Sonic Hedgehog (SHH; P<0.05), PTCH1 (P<0.05) and GLI effectors (GLI1 P<0.05, GLI2 P<0.0001), which were reduced by C2CD3 knockdown by C2CD3-sequence-specific LNA-GapmeRs.
Conclusion: Our results suggest DKD is associated with upregulation of C2CD3, and driven by upregulated TGF-β1. In vitro studies of knockdown of C2CD3 in presence of TGF-β1 abrogated aberrant pathological responses in proximal tubules and mechanistic studies support view that C2CD3 signals via HHS-pathway. These studies suggest dysregulation of cilial function may be novel mechanism involved in development of DKD.
Dr Sanela Redzepagic is a nephrologist. She is a PhD candidate at the Faculty of Medicine and Health at the University of Sydney, with Prof. Pollock’s renal research group, Kolling Institute of Medical Research, The Royal North Shore Hospital (RNSH), Sydney and under supervision by Prof. Victoria Cogger. Dr Sanela Redzepagic was awarded the APA (RTP) scholarship and a supplementary stipend from the Ramsay Health Care Foundation.
Her PhD study focuses on investigating a role of a novel mechanism of long non-coding RNAs (lncRNAs) in diabetic kidney disease – investigating a novel antisense lncRNA Gm10603 and its relationship to its flanking protein coding genes, particularly C2CD3, a primary cilia gene.
Dr Redzepagic completed her undergraduate medical degree BSc (Med) MBBS at the University of New South Wales (UNSW). She completed her nephrology training at the Royal Prince Alfred Hospital (RPAH) and the East Coast Renal Networks and was awarded the FRACP with subspecialty in nephrology (December 2015).