2061

Targeting N-RasGTPase in Murine Proximal Tubule Epithelial Cells and its Potential for a Therapeutic Strategy in a Murine Model of Tubulointerstitial Fibrosis.

*SubashSomalankaClaire C. Sharpe**, Mysore K Phanish* and Mark E Dockrell*.

*South West Thames Institute for Renal Research, St Helier Hospital, Carshalton, Surrey, UK and ** Department of Renal Medicine, King’s College, London, UK

Introduction: Tubulo-interstitial fibrosis (TIF) is a hallmark of progressive chronic kidney disease and recent evidence suggests that proximal tubule epithelial cell(PTEC) injury leads to TIF and glomerulosclerosis. Connective tissue growth factor (CTGF) and TGF1 are critical factors in Fibrosis. Ras GTPases (H-Ras, KA-Ras, KB-Ras & N-Ras) are small monomeric enzymatic proteins, which in a GTP bound active state regulate downstream cell signalling pathways. Previous studies in both human renal diseases and murine models of nephropathy have shown that there is altered expression of Ras isoforms. Anti-Sense Oligonucleotides (ASO) are an effective way of targeting Ras GTPases as shown previously by our group in a murine models of renal fibrosis. Recently, Wang et alshowed that specific targeting of K-Ras has ameliorated renal fibrosis in a rat model of unilateral ureteric obstruction. Our grouphad previously published in in vitro studies of human PTECs that induction of CTGF and autoinduction of TGF1 are dependent on N-Ras, however, Hendry et al had published that Ras GTPases have distinct functions in the control of proliferation and apoptosis in mouse and human mesangial cells. Hence, weinvestigatedour previously published work in murine Proximal Tubule Epithelial Cells (mPTEC) with a view to translate the work in vivo.

Methods:Six ASOs targeting N-Ras were provided by ISIS Pharmaceuticals, California. In vitro - Cells derived from S3 segment of a proximal tubule from a SV40 transformed brinster transgenic mouse were used. Serum starved cells were treated with TGF1[2.5ng/ml; 24hrs]. Transfection of ASO (200nM) was achieved by using Lipofectamine LTX in presence of OptiMEM I for 24 hrs. Negativecontrol ASO, positive control ASO targeting PTEN gene and ASOs targeting N-Ras gene were screened. Specificity & efficacy of knockdown was assessed by qPCR. Toxicity was assessed by LDH assay. In vivo – CD1 male outbred mice were injected on Day1 and Day 21 with vehicle (V, normal saline) versus folic acid (FA, 125mg/kg, IV) to induce chronic folic acid induced nephropathy (FAIN) at Day 84. Serum samples were analyzed by Cystatin C ELISA and BUN assay. RNA extractedfrom kidneys was probed for CTGF and TGF1. Paraffin sections of kidneys were stained with H&E, Trichrome and Picro-Sirius Red. TIF was scored by a histopathologist in a blinded manner.ASO were screened in healthy mice to assess their efficacy and toxicity. Efficacy of N-Ras knockdown in kidneys was assessed by qPCR. Serum samples were assessed for toxicity by transaminase and BUN assay.

Results:In vitro - TGF1 treatment for 24 hrs significantly increased the expression of N-Ras (p<0.001) and K-Ras (p<0.001). TGF1 induced CTGF mRNA (p<0.01) and auto-induced TGF1(p<0.05) at 24 hrs. ASO3 & ASO5 transfection with Lipofectamine LTX provided the best knockdown (60-75%) of N-Ras RNA. ASO3 & ASO5 significantly inhibited TGF1-driven induction of CTGF and autoinduction of TGF1. In vivo – Cortical TIF(10 – 15 %) was observed in the CD-1 mice after day84 with increased expression of CTGF and TGF1 mRNA. Impaired renal function at Day 84 was assessed by BUN (p=0.056) and Cystatin C ELISA (p=0.02)for V versus FA groups. ASO screening showed a significant knockdown (p<0.001) of N-Ras RNA. BUN and transaminase assay showed that they were safe to be used in vivo. ASO3 & ASO4 were selected to be used in FAIN.

Conclusions:Our results show TGF1-driven induction of CTGF and autoinduction of TGF1 is dependent on N-Ras which supports our group’s previous work in human PTECs.We also showed for the first time that TGF1 significantly increased the expression of N-ras in mPTEC. FAIN is a suitable model for studying TIF and shows overexpression of CTGF and TGF1. We have shown that ASOs can be safely used to target N-Ras in vivo. ASO administration in FAIN is ongoing. We believe targeting N-Ras in FAIN will establish it as a potential target for future therapy in human TIF.