S REDZEPAGIC1, C HUANG1, C POLLOCK1, U PANCHAPAKESAN1
1Renal Research Group, Kolling Institute, Royal North Shore Hospital, University of Sydney, Sydney, Australia
Aim: To assess if primary cilial Protein#1 is regulated by transforming growth factor beta (TGFB1) and evaluate if gene-silencing with sequence-specific LNA GapmeRs (Exiqon) affect downstream markers of kidney fibrosis.
Background: Primary cilia on kidney proximal tubule cells (PTCs) are sensitive to luminal mechanosensory stimuli and are a novel pathway linking luminal changes with tubular dysfunction. Our target, primary cilia Protein#1, is integral to the hedgehog signalling pathway, hence relevant to kidney fibrosis.
Methods: Primary cultures of mice PTC and human kidney (HK2) cells were transfected using control and Protein#1-sequence-specific LNA GapmeRs. Mice PTCs and HK2 cells were exposed to 5 or 2 ng/ml TGFB1 respectively for 48h. Kidney tissue from control and mice with streptozotocin-induced diabetes mellitus was harvested. mRNA expression of Protein#1 and downstream profibrotic markers were assessed.
Results: Protein#1 was upregulated in kidneys of diabetic mice (N=10-12,P<0.001) and HK2 cells (N=4,P<0.05) exposed to TGFB1 for 48h. A similar trend was seen in mice PTCs exposed to TGFB (N=4,P=0.08).Mice PTCs exposed to TGFB upregulated fibronectin (N=4,P<0.05) and collagen1 (N=3,P<0.05). Silencing of Protein#1 using sequence-specific LNA GapmeRs reduced expression of collagen1 (N=3,P<0.05), with a similar trend observed for fibronectin (N=4,P<0.06).HK2 cells exposed to TGFB upregulated collagen 4 (N=4,P<0.05), plasminogen activator inhibitor (PAI) (N=4,P=0.01) with a similar trend observed for fibronectin (N=4,P=0.08). Silencing of Protein#1-as above reduced fibronectin (N=4,P<0.01) and PAI (N=4,P<0.001). A similar trend was observed for collagen 4 expression (N=4,P=0.06).
Conclusion: Primary cilia Protein#1 is upregulated in mice diabetic kidneys and in TGFB1-exposed HK2 cells. Gene-silencing with Protein#1-sequence-specific LNA GapmeR reduced downstream markers of kidney fibrosis, suggesting its involvement in kidney fibrosis thus suggesting Protein#1 is a potential therapeutic target.
Sanela Redzepagic is a nephrologist from Sydney. She is undertaking a PhD candidature at The University of Sydney, with Prof. Pollock’s Renal Research Lab, Koling Institute – Royal North Shore Hospital, to investigate a role of LNCRNA in the DKD